ES2956757T3 - Accurate molecular barcode coding - Google Patents

Abstract

According to some embodiments herein, compositions and methods for accurate barcoding of nucleic acids are described. The compositions and methods involve a plurality of unique oligonucleotide species comprising unique molecular barcodes. Molecular barcodes have a relatively low G (guanine) content, for example, each of the barcodes contains less than 50% guanine or the total set of barcodes contains on average less than 12.5% of guanine. The barcode array is intended to show reduced bias in amplification and analysis. (Automatic translation with Google Translate, without legal value)

Description

DESCRIPTION

Accurate molecular barcode coding

FIELD

Embodiments herein generally relate to compositions and methods for precise barcoding of molecules, for example nucleic acid molecules.

WO02059355 describes a family of minimally cross-hybridizing nucleotide sequences and methods of their use. In particular, a specific family of 1168 24mers is described. WO2015031691 describes methods, compositions and kits for multiplex analysis of nucleic acids from single cells. The methods, compositions and systems can be used for massively parallel sequencing of single cells. US2010159533 describes methods and kits for the selective preparation of cDNA relatively free of sequences found in rRNA and subcellular RNAs. The methods and kits use approximately 200 hexamer sequences that target messenger RNA. BUSCHMANN T et al. (BMC BIOINFORMATICS, 2014, vol. 15(1):264) describes improving the detection of barcoded reads in high-throughput DNA sequencing data by controlling the false discovery rate. WO2016138500 describes methods and compositions for labeling a nucleic acid in a sample with a stochastic barcode using an extendable oligonucleotide with a universal priming site and a target-specific region. Some embodiments relate to methods and compositions for characterizing a sample by identifying the alpha chain or beta chain of the TCR of a T cell. WO2015134787 describes methods for amplification bias correction and quantification of adaptive immune cells in a sample using templates. synthetics that include random oligonucleotide sequences.

SUMMARY

The invention is set forth in the attached set of claims.

The invention provides a composition comprising at least 1000 unique oligonucleotide species, each unique oligonucleotide species comprising a barcode region and a uniform region, the barcode region comprising a molecular barcode comprising at least 7 nucleotides, wherein the uniform region is 3' of the barcode region, the uniform region comprising a target-specific region 3' of the barcode region, the target-specific region comprising at least 10 complementary nucleotides to a target nucleic acid, wherein the single oligonucleotide species comprise different nucleic acid sequences in their barcode regions, and wherein: (a) the composition consists essentially of single oligonucleotide species wherein each barcode Molecule bars have a G content of less than 50%; and/or (b) the molecule barcodes of all unique oligonucleotide species in the composition collectively have a G content of no more than 12.5%, wherein the barcode region further comprises a G code. Sample bars comprising at least 3 nucleotides. In some embodiments, the composition consists essentially of single oligonucleotide species where each molecular barcode has a G content of less than 50%. In some embodiments, the molecular barcodes of all unique oligonucleotide species of the composition collectively have a G content of no more than 12.5%. In some embodiments, the unique oligonucleotide species are arranged in at least two spatially isolated groups, each group comprising at least 100 unique oligonucleotides of the unique oligonucleotide species, wherein the unique oligonucleotides of the same group comprise the same code sequence. sample barcodes, and where different unique oligonucleotides from the same group comprise different molecule barcode sequences. In some embodiments, the sample barcode of each single oligonucleotide species has a G content of 50% or less. In some embodiments, the barcode region of each single oligonucleotide species has a G content of 50% or less. In some embodiments, the barcodes of the single oligonucleotide species molecules collectively have a G content of less than 12.5%. In some embodiments, the barcode regions of the single oligonucleotide species collectively have a G content of no more than 12.5%. In some embodiments, for at least 95% of the unique oligonucleotide species, any G in the molecular barcode is not adjacent to another G. In some embodiments, the composition consists essentially of unique oligonucleotide species for which any G in the molecular barcode is not adjacent to another G. In some embodiments, at least 95% of the molecule barcodes of the unique oligonucleotide species comprise a sequence that sums to at least 6 alternating H and N , where each "H" is any one of A, C or T, and where each "N" is any one of A, G, C or T. In some embodiments, at least 95% of the codes of Molecular bars of the single oligonucleotide species comprise the sequence HNHNHNHN, where each "H" is any one of A, C or T, and where each "N" is any one of A, G, C or T. In some embodiments, each molecular barcode of the unique oligonucleotide species comprises the sequence HNHNHNHN, where each "H" is any one of A, C, or T, and where each "N" is any one of A, G, C, or T. In some embodiments, each molecular barcode of the unique oligonucleotide species comprises the sequence HHHHHHHH, where each "H" is a any of A, C, or T. In some embodiments, each of the single oligonucleotide species comprises a spacer 3' of the barcode region and 5' of the target-specific region, said spacer comprising the sequence HNHNHNHN, wherein each "H" is any one of A, C or T, and where each "N" is any one of A, G, C or T. In some embodiments, each of the single oligonucleotide species comprises a spacer 3' of the barcode region and 5' of the target-specific region, said spacer comprising the sequence HHHHHHHH, where each "H" is any one of A, C or T. In some embodiments, the specific region The target comprises an oligo dT sequence. In some embodiments, for each unique oligonucleotide species, the molecular barcode is 3' to the sample barcode. In some embodiments, for each unique oligonucleotide species, the sample barcode is 3' to the molecular barcode. In some embodiments, each oligonucleotide species has a length of at least 24 nucleotides. In some embodiments, each oligonucleotide species is 24-140 nucleotides in length. In some embodiments, the composition comprises at least 6,500 unique oligonucleotide species. In some embodiments, the composition comprises at least 65,000 unique oligonucleotide species. In some embodiments, the composition comprises at least two oligonucleotides of the same single oligonucleotide species. In some embodiments, no two oligonucleotides of the composition are of the same single oligonucleotide species. In some embodiments, the composition comprises at least 48 groups. In some embodiments, the unique oligonucleotide species of each group are immobilized on a substrate, such that the sample barcodes but not the molecular barcodes are the same for the oligonucleotide species immobilized on each substrate. In some embodiments, the substrate comprises a discrete region of a surface, such that the surface may comprise two or more substrates. In some embodiments, the substrate comprises a bead. In some embodiments, each of the single oligonucleotide species further comprises an adapter configured to immobilize the single oligonucleotide to the substrate, wherein said barcode region is located 3' of the adapter. In some embodiments, the uniform region comprises a target-specific region comprising a sequence flanking an immune cell receptor or immunoglobulin variable region coding sequence. In some embodiments, the immune cell receptor variable region coding sequence is a T cell receptor variable region coding sequence, a B cell receptor variable region coding sequence, or a combination thereof. In some embodiments, the invention provides a kit comprising the composition of the invention, wherein the target-specific region comprises a sequence flanking an immune cell receptor or immunoglobulin variable region coding sequence, and a primer configured to hybridize in an opposite side of the variable region as the target-specific region and to hybridize to a strand complementary to a strand hybridized by the target-specific region, such that the primer is configured to amplify the variable region together with the target-specific region. aim. In some embodiments, the primer and target-specific region are configured to amplify a nucleic acid of at least 1 kb and comprising the variable region. In some embodiments, the kit primer forms part of the composition comprising the single oligonucleotide species. In some embodiments, the kit primer is part of another composition that is separate from the composition comprising the single oligonucleotide species.

The invention also provides a method of specifically barcoding a plurality of nucleic acids from two or more samples, each sample comprising nucleic acids, comprising contacting each sample with a pool comprising at least 100 species of unique oligonucleotides. , wherein each sample is contacted in spatial isolation from the other samples, each unique oligonucleotide species comprising a barcode region comprising: a molecular barcode comprising at least 7 nucleotides; a sample barcode comprising at least 3 nucleotides; and a uniform region 3' of the barcode region, the uniform region comprising a target-specific region 3' of the barcode region, the target-specific region comprising at least 10 nucleotides complementary to a target nucleic acid ; wherein the unique polynucleotide species of each group comprise the same sample barcode, and comprise different molecular barcodes, and wherein: (a) the unique oligonucleotide species contacted with the sample essentially consist of species of single oligonucleotides wherein each molecular barcode has a G content of less than 50%; and/or (b) the molecular barcodes of all single oligonucleotide species collectively have a G content of no more than 12.5%; hybridizing target-specific regions of at least some oligonucleotides of the single oligonucleotide species to at least some of the sample nucleic acids; and extending the hybridized oligonucleotides, thereby producing chains comprising an oligonucleotide of the unique oligonucleotide species and a sequence complementary to the target, wherein for each sample, the chains comprise the same sample barcode and different molecular barcodes , and where for different samples, the molecular barcodes are different. In some embodiments, the single oligonucleotide species contacted with the sample essentially consist of single oligonucleotide species where each molecular barcode has a G content of less than 50%. In some embodiments, the molecular barcodes of all single oligonucleotide species collectively have a G content of no more than 12.5%. In some embodiments, the method further comprises determining the nucleic acid sequences of the chains comprising the oligonucleotides of the single oligonucleotide species and the sequence complementary to the target. In some embodiments, the at least 100 unique oligonucleotide species of each group are immobilized on a substrate, such that the unique oligonucleotide species immobilized on a given substrate comprise the same barcode. sample, and different species of single oligonucleotides immobilized on the substrate comprise different molecular barcodes. In some embodiments, each sample barcode has a G content of 50% or less. In some embodiments, the molecular barcodes of the single oligonucleotide species collectively have a G content of less than 12.5%. In some embodiments, the barcode regions of the single oligonucleotide species collectively have a G content of no more than 12.5%. In some embodiments, for at least 95% of the unique oligonucleotide species, any G in the molecular barcode is not adjacent to another G. In some embodiments, each group consists essentially of unique oligonucleotide species for which any G in the molecular barcode is not adjacent to another G. In some embodiments, at least 95% of the molecular barcodes of the unique oligonucleotide species comprise a sequence that sums to at least 6 alternating H and N, wherein each "H" is any one of A, C or T, and where each "N" is any one of A, G, C or T. In some embodiments, at least 95% of the barcodes Molecular elements of the single oligonucleotide species comprise the sequence HNHNHNHN, where each "H" is any one of A, C or T, and where each "N" is any one of A, G, C or T. In some embodiments , each molecular barcode of the unique oligonucleotide species comprises the sequence HNHNHNHN, where each "H" is any one of A, C or T, and where each "N" is any one of A, G, C or T. In some embodiments, each molecular barcode of the unique oligonucleotides comprises the sequence HHHHHHHH, where each "H" is any one of A, C or T. In some embodiments, each unique oligonucleotide comprises a 3' spacer of the barcode region and 5' of the target-specific region, said spacer comprising the sequence HNHNHNHN, where each "H" is any one of A, C, or T, and where each "N" is any one of A, G, C, or T. In some embodiments, each unique oligonucleotide comprises a spacer 3' of the barcode region and 5' of the target-specific region, said spacer comprising the sequence HHHHHHHH, wherein each "H " is any one of A, C, or T. In some embodiments, at least one group comprises at least two oligonucleotides of the same single oligonucleotide species. In some embodiments, no group comprises two oligonucleotides of the same single oligonucleotide species. In some embodiments, the target-specific region comprises an oligo dT sequence. In some embodiments, for each unique oligonucleotide species, the molecular barcode is 3' to the sample barcode. In some embodiments, for each unique oligonucleotide species, the sample barcode is 3' to the molecular barcode. In some embodiments, each unique oligonucleotide species has a length of at least 24 nucleotides. In some embodiments, each unique oligonucleotide species has a length of 24-140 nucleotides. In some embodiments, each pool comprises at least 6,500 unique oligonucleotide species. In some embodiments, each pool comprises at least 65,000 unique oligonucleotide species. In some embodiments, at least 48 unique samples are contacted with a single group. In some embodiments, at least 99% of the samples comprise no more than one cell each. In some embodiments, the unique oligonucleotide species of each group are immobilized on a substrate, such that the sample barcodes but not the molecular barcodes are the same for the unique oligonucleotide species immobilized on each substrate. the plurality. In some embodiments, the substrate comprises a spatially isolated region of a surface, such that substrates of different groups comprise different spatially isolated regions of the surface. In some embodiments, the substrate comprises a bead. In some embodiments, each of the single oligonucleotide species further comprises an adapter configured to immobilize the single oligonucleotide to the substrate, wherein said barcode region is located 3' of the adapter. In some embodiments, the uniform region comprises a target-specific region comprising a sequence flanking a sequence encoding the variable region of an immune cell receptor or immunoglobulin. In some embodiments, the variable region is from a T cell receptor or a B cell receptor, or a combination thereof. In some embodiments, the method further comprises contacting extended chains comprising an oligonucleotide of the single oligonucleotide species and a sequence complementary to the target with primer configured to hybridize on an opposite side of the variable region as the target-specific region, and to hybridize with a strand complementary to a strand hybridized by the specific region of the target. As such, the method may comprise amplifying sequences that encode variable regions of a T cell receptor, B cell receptor or immunoglobulin. In some embodiments, the method amplifies a sequence of at least 1 kb, comprising the variable region coding sequence.

The invention also provides a method of making a composition comprising single oligonucleotides comprising: providing a plurality of different sample barcodes comprising at least 3 nucleotides each, providing a plurality of different molecular barcodes comprising at least 7 nucleotides each; synthesizing a plurality of unique oligonucleotide species, each unique oligonucleotide species comprising a barcode region comprising a sample barcode and a molecular barcode; and a uniform region 3' of the barcode region, the uniform region comprising a target-specific region 3' of the barcode region, the target-specific region comprising at least 10 nucleotides complementary to a target nucleic acid ; and arranging the unique oligonucleotide species in spatially isolated groups, wherein each group comprises multiple unique oligonucleotide species, such that the unique oligonucleotide species of the same group comprise the same sample barcode sequence, and wherein different species of unique oligonucleotides of the same group comprise different molecular barcode sequences, and wherein each group comprises at least 1000 species of unique oligonucleotides, in which that for each group (a) the plurality of single oligonucleotide species consists essentially of single oligonucleotide species wherein each molecular barcode has a G content of less than 50%; and/or b) the molecular barcodes of all unique oligonucleotide species of the plurality collectively have a G content of no more than 12.5%. In some embodiments, the plurality of single oligonucleotide species essentially consists of single oligonucleotide species wherein each molecular barcode has a G content of less than 50%. In some embodiments, the molecular barcodes of all of the plurality of unique oligonucleotide species collectively have a G content of no more than 12.5%. In some embodiments, each molecular barcode has a G content of 50% or less. In some embodiments, each sample barcode has a G content of 50% or less. In some embodiments, the sample barcodes of the single oligonucleotide species collectively have a G content of no more than 12.5%. In some embodiments, the molecular barcodes of the single oligonucleotide species collectively have a G content of less than 12.5%. In some embodiments, the barcode regions of the single oligonucleotide species collectively have a G content of no more than 12.5%. In some embodiments, for at least 95% of the single oligonucleotide species, any G in the molecular barcode is not adjacent to another G. In some embodiments, the plurality of single oligonucleotide species consists essentially of species of unique oligonucleotides for which any G in the molecular barcode is not adjacent to another G. In some embodiments, at least 95% of the molecular barcodes of the unique oligonucleotide species comprise a sequence totaling at least 6 alternating H and N, where each "H" is any one of A, C or T, and where each "N" is any one of A, G, C or T. In some embodiments, at least 95 % of the molecular barcodes of the unique oligonucleotides comprise the sequence HNHNHNHN, where each "H" is any one of A, C or T, and where each "N" is any one of A, G, C or T In some embodiments, each molecular barcode of the unique oligonucleotides comprises the sequence HNHNHNHN, where each "H" is any one of A, C or T, and where each "N" is any one of A, G, C or T. In some embodiments, each molecular barcode of the unique oligonucleotides comprises the sequence HHHHHHHH, where each "H" is any one of A, C or T. In some embodiments, each unique oligonucleotide species comprises a spacer 3' of the barcode region and 5' of the target-specific region, said spacer comprising the sequence HNHNHNHN, where each "H" is any one of A, C, or T, and where each "N" is any one of A, G, C, or T. In some embodiments, each single oligonucleotide species comprises a spacer 3' of the barcode region and 5' of the target-specific region, said spacer comprising the sequence HHHHHHHH , where each "H" is any one of A, C or T. In some embodiments, the target-specific region comprises an oligo dT sequence. In some embodiments, for each unique oligonucleotide species, the molecular barcode is 3' to the sample barcode. In some embodiments, for each unique oligonucleotide species, the sample barcode is 3' to the molecular barcode. In some embodiments, each unique oligonucleotide species has a length of at least 24 nucleotides. In some embodiments, each unique oligonucleotide species has a length of 24-140 nucleotides. In some embodiments, each pool comprises at least 6,500 unique oligonucleotide species. In some embodiments, each pool comprises at least 65,000 unique oligonucleotide species. In some embodiments, at least 48 groups are made. In some embodiments, the method further comprises immobilizing the unique oligonucleotide species of each group on a substrate, such that the sample barcodes but not the molecular barcodes are the same for the oligonucleotide species immobilized on each substrate of plurality. In some embodiments, the substrates comprise discrete regions of a surface. In some embodiments, the substrates comprise beads. In some embodiments, the single oligonucleotide species are arranged in spatially isolated groups simultaneously with said synthesis. In some embodiments, the single oligonucleotide species are arranged in spatially isolated groups after said synthesis. In some embodiments, the uniform region comprises a target-specific region comprising a sequence flanking a variable region coding sequence of an immune cell receptor or immunoglobulin. In some embodiments, the immune cell receptor variable region coding sequence is a T cell receptor variable region coding sequence, a B cell receptor variable region coding sequence, or a combination thereof. In some embodiments, the kit further comprises a primer configured to hybridize to an opposite side of the variable region coding sequence as the target-specific region, and to hybridize to a strand complementary to a strand hybridized by the target-specific region, and It is therefore configured, together with the target-specific region, to amplify the variable region coding sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic diagram illustrating an oligonucleotide species comprising a barcode region 5' of a uniform region, according to some embodiments herein.

FIG. 1B is a schematic diagram illustrating an oligonucleotide species comprising a 5' barcode region of an oligo dT sequence, according to some embodiments herein.

FIG. 1C is a schematic diagram illustrating an oligonucleotide species comprising a barcode region 5' of a specific region of a gene, according to some embodiments herein. FIG. 2A is a schematic diagram illustrating a barcode region comprising a molecular barcode 5' of a sample barcode, and which may comprise a barcode region of a single oligonucleotide species according to some embodiments herein.

FIG. 2B is a schematic diagram illustrating a barcode region comprising a 5' sample barcode of a molecular barcode, and which may comprise a barcode region of a single oligonucleotide species according to some realizations hereof.

FIGS. 3A-3H are diagrams illustrating a variety of oligonucleotide species configurations according to some embodiments herein. FIG. 3A illustrates a reference oligonucleotide. FIG. 3B illustrates an oligonucleotide with a base spacer 3' of the barcode region. FIG. 3C illustrates an oligonucleotide with a 5-base spacer 3' of the barcode region. FIG. 3D illustrates an oligonucleotide in which the positions of the molecular barcode (denoted as "NNNNNNNNNN") and the sample barcode within the barcode region are swapped compared to FIG.

3A, such that the molecular barcode is 5' from the sample barcode. FIG. 3E illustrates an oligonucleotide in which the positions of the molecular barcode (denoted as "NNNNNNNNNN") and the sample barcode within the barcode region are swapped compared to FIG. 3A, such that the molecular barcode is 5' from the sample barcode, and includes a 3-base spacer. FIG. 3F illustrates an oligonucleotide in which the positions of the molecular barcode (denoted as "NNNNNNNNNN") and the sample barcode within the barcode region are swapped, such that the molecular barcode is 5' of the sample barcode, and includes a 5-base spacer. FIG. 3G illustrates an oligonucleotide in which the molecular barcode comprises the sequence HHHHHHHH (in which each H is an A, C or T, and in which each H can be the same or different from any other H). FIG. 3H illustrates an oligonucleotide in which the molecular barcode comprises the sequence HNHNHNHN (in which each H is an A, C or T, and in which each H can be the same or different from any other H).

FIG. 4A is a diagram illustrating the use of nucleotides in a sampling of single conventional oligonucleotide species.

FIG. 4B is a diagram illustrating the use of nucleotides in a sampling of single conventional oligonucleotide species.

FIG. 4C is a diagram illustrating the use of nucleotides in a sampling of single conventional oligonucleotide species.

FIG. 5A is a diagram illustrating nucleic acid sequences of oligonucleotide species that were constructed according to some embodiments herein. The SEQ ID NO: indicated in the rightmost columns of FIGS 5A-5D refer to the polynucleotide sequence that includes the "anchor", "1° be", "2° be", "Spacer" (if there is), and "Recognition Sequence 5 to 3' " as shown. Each "anchor" of FIGS. 5A-5D has the polynucleotide sequence of SEQ ID NO: 97.

FIG. 5B is a diagram illustrating nucleic acid sequences of oligonucleotide species that were constructed according to some embodiments herein.

FIG. 5C is a diagram illustrating nucleic acid sequences of oligonucleotide species that were constructed according to some embodiments herein.

FIG. 5D is a diagram illustrating nucleic acid sequences of oligonucleotide species that were constructed according to some embodiments herein.

FIG. 6 is a per-individual error correction graph for an analysis using single oligonucleotide species according to some of the embodiments herein.

FIG. 7 is a filter and individual MI crossover error correction graph for an analysis using single oligonucleotide species according to some embodiments herein.

FIG. 8 is a diagram that schematically illustrates, without being limited by any theory, the possible amplification errors that can result from oligonucleotides with high G contents.

DETAILED DESCRIPTION

According to some embodiments herein, methods and compositions for barcoding and analysis of precise nucleic acids are described. In some embodiments, individual nucleic acids in a sample may be associated with a unique barcode (e.g., a "molecular barcode"), such that upon amplification and sequence analysis, the individual nucleic acids of a sample can be quantified. Without being limited by theory, it is contemplated that bias favoring or disfavoring the representation, amplification or properties of certain types of barcode sequences may interfere with the quantification and analysis of individual nucleic acids in a sample (possible sources of bias in some amplification events are illustrated schematically in FIG. 8). According to some embodiments herein, configurations and characteristics of single oligonucleotide species comprising barcodes are described, wherein the single oligonucleotide species are configured to minimize barcode-related bias and produce accurate analysis. of nucleic acids. Without being limited by any theory, it is contemplated that barcode regions comprising features such as a guanosine (G) content of less than 50%, and/or without two consecutive "G"s in the barcode region may minimize bias that could otherwise confound the quantification and/or analysis of nucleic acids from a sample. Optionally, individual nucleic acid molecules from a given sample may also be associated with a “sample barcode,” such that nucleic acids associated with the barcode can subsequently be grouped together for efficient batch analysis of nucleic acids. of two or more samples, for example through next generation sequencing.

Nucleic adjuvants

Various nucleic acids are described in accordance with some embodiments herein. For example, the oligonucleotide species, samples and/or targets may comprise nucleic acids.

As used herein, a "nucleic acid" refers to a polynucleotide sequence, or fragment thereof. A nucleic acid may comprise nucleotides. A nucleic acid can be exogenous or endogenous to a cell. A nucleic acid can exist in a cell-free environment. A nucleic acid may comprise, consist of, or consist essentially of a gene or fragment thereof. A nucleic acid may comprise, consist of, or consist essentially of DNA. A nucleic acid may comprise, consist of, or consist essentially of RNA. A nucleic acid may comprise one or more analogues (for example, an altered backbone, sugar or nucleobase). Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycolic nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, florophores (e.g. rhodamine or sugar-linked flurescein), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogues, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine and wiosin. "Nucleic acid", "polynucleotide", "target polynucleotide" and "target nucleic acid" can be used interchangeably.

As used herein, "upstream" (and variations of this root term) refers to a position that is relatively 5' in a nucleic acid (e.g., 5' compared to the reference position). As used herein, "downstream" (and variations of this root term) refers to a position that is relatively 3' in a nucleic acid (e.g., 3' compared to the reference position). For example, as shown in FIG. 2A, the "sample barcode" is 3' from the "molecular barcode" and is understood to be downstream of the molecule "barcode." For example, as shown in FIG. 2B, the "sample barcode" is 5' from the "molecular barcode" and is understood to be upstream of the molecule "barcode".

A nucleic acid may comprise one or more modifications (eg, a base modification, a backbone modification), to provide the nucleic acid with a new or improved characteristic (eg, improved stability). A nucleic acid may comprise a nucleic acid affinity tag. A nucleoside may comprise, consist of, or consist essentially of a base-sugar combination. The base portion of the nucleoside may be a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. Nucleotides may comprise, consist of, or consist essentially of nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group may be linked to the 2', 3' or 5' hydroxyl moiety of the sugar. When forming nucleic acids, phosphate groups can covalently link adjacent nucleosides to each other to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound; however, linear compounds are generally suitable. Furthermore, linear compounds can have internal complementarity of nucleotide bases and can therefore fold in a way that produces a fully or partially double-stranded compound. Within nucleic acids, phosphate groups can be commonly referred to as forming the internucleoside backbone of the nucleic acid. The linkage or backbone of the nucleic acid may be a 3' to 5' phosphodiester bond.

A nucleic acid may comprise a modified backbone and/or modified internucleoside linkages. Modified backbones may include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Suitable modified nucleic acid backbones containing a phosphorus atom may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl phosphonates and other alkyls, such as 3'-alkylene phosphonates, 5'-phosphonates. -alkylene, chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranephosphates having normal 3'-5' bonds, analogs with 2'-5' bonds and those that have inverted polarity where one or more internucleotide bonds are 3' to 3', 5' to 5' or 2' to 2' bonds.

A nucleic acid may comprise polynucleotide backbones formed by short-chain alkyl or cycloalkyl internucleoside bonds, heteroatom and mixed alkyl or cycloalkyl internucleoside bonds, or one or more short-chain heteroatomic or heterocyclic internucleoside bonds. These may include those with morpholino bonds (formed in part from the sugar portion of a nucleoside); siloxane backbones; main sulfide, sulfoxide and sulfone structures; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; main structures of amide; and others that have mixed component parts of N, O, S and CH ₂ .

A nucleic acid may comprise a nucleic acid mimetic. The term "mimetic" includes, for example, polynucleotides in which only the furanose ring or both the furanose ring and the internucleotide bond are replaced by non-furanose groups; Substitution of only the furanose ring can also be called a sugar substitute. The heterocyclic base moiety or a modified heterocyclic base moiety may be maintained for hybridization with an appropriate target nucleic acid. One of these nucleic acids may be a peptide nucleic acid (PNA). In a PNA, the sugar backbone of a polynucleotide may be replaced by an amide-containing backbone, in particular an aminoethylglycine backbone. The nucleotides can be conserved and attached directly or indirectly to the aza nitrogen atoms of the amide portion of the backbone. The backbone of ANP compounds may comprise two or more linked aminoethylglycine units, giving ANP an amide-containing backbone. Heterocyclic base molecules can bind directly or indirectly to the aza nitrogen atoms of the amide portion of the backbone.

A nucleic acid may comprise a morpholino backbone. For example, a nucleic acid may comprise a 6-membered morpholino ring instead of a ribose ring. In some of these embodiments, a phosphorodiamidate or other non-phosphodiester internucleoside bond may substitute for a phosphodiester bond.

A nucleic acid may comprise linked morpholino units (i.e., morpholino nucleic acid) having heterocyclic bases attached to the morpholino ring. The linking groups can link the monomeric morpholino units into a morpholino nucleic acid. Morpholino-based nonionic oligomeric compounds may have fewer unwanted interactions with cellular proteins. Morpholino-based polynucleotides can be nonionic nucleic acid mimics. A variety of compounds within the morpholino class can be linked using different linking groups. An additional class of mimetic polynucleotides can be called cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in a nucleic acid molecule can be replaced by a cyclohexenyl ring. DMT-protected phosphoramidite monomers of CeNA can be prepared and used for the synthesis of oligomeric compounds by phosphoramidite chemistry. Incorporation of CeNA monomers into a nucleic acid strand can increase the stability ^of a D ⁿ /RNA hybrid. CeNA oligoadenylates can form complexes with nucleic acid complements with stability similar to that of native complexes. Another modification may include locked nucleic acids (LNA) in which the 2'-hydroxyl group is bonded to the 4' carbon atom of the sugar ring thereby forming a 2'-C,4'-C-oxymethylene bond forming thus a bicyclic sugar fraction. The bond may comprise, consist of, or consist essentially of a methylene group (-CH ₂ -), linking the 2' oxygen atom and the 4' carbon atom where n is 1 or 2. LNA and analogs of LNA can show very high duplex thermal stabilities with the complementary nucleic acid (Tm=+3 at 10° C), stability against 3'-exonucleolytic degradation and good solubility properties.

In some embodiments, a nucleic acid may also include nucleobase modifications or substitutions (often referred to simply as "bases"). As used herein, "unmodified" or "natural" nucleobases may include purine bases (e.g., adenine (A) and guanine (G)), and pyrimidine bases (e.g., thymine (T), cytosine (C) and uracil (U)). Modified nucleobases may include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases , 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5 -halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7 -deazaadenine and 3-deazaguanine and 3-deazaadenine. Modified nucleobases may include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)( 1,4)benzothiazin-2(3H)-one), G-clamps as a substituted cytidine phenoxazine (e.g. 9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin- 2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (Hpyrido(3',':4,5)pyrrolo[2,3-d] pyrimidin-2-one).

Samples

As used herein, the term "sample" refers to a composition comprising targets. Samples suitable for analysis by the disclosed methods, devices and systems include, but are not limited to, cells, single cells, tissues, organs or organisms. In some embodiments, a sample comprises raw or unprocessed samples, for example, a whole cell, a whole population of cells, or an entire tissue. In some embodiments, a sample comprises an isolated cell or cell extract, or a nucleic acid-containing fraction thereof, for example isolated nucleic acids, or a composition comprising enriched or isolated nucleic acids. In some embodiments, a sample comprises a tissue fixed, a cell or a fraction containing nucleic acids thereof. In some embodiments, a sample comprises a frozen tissue, cell, or nucleic acid-containing fraction thereof. In some embodiments, a sample comprises a solution containing nucleic acids. In some embodiments, a sample comprises a solution comprising nucleic acids. In some embodiments, a sample comprises nucleic acids in a solid format, for example, lyophilized nucleic acids and the like.

Unique oligonucleotide species

As used in the compositions, methods and oligonucleotides according to some embodiments herein, a "single oligonucleotide species" refers to an oligonucleotide, for example DNA or RNA, that has a sequence that differs by at least one base. of another single oligonucleotide species. The single oligonucleotide species of a composition according to some embodiments herein may share certain structural characteristics or formats, but may have nucleic acid sequences different from each other. Single oligonucleotide species can be single-stranded or double-stranded. A composition may comprise a variety of 100, 1000, 6500 or 65,000 species of unique oligonucleotides. Optionally, the composition comprising single oligonucleotide species may also comprise two or more oligonucleotides of the same single oligonucleotide species. By way of example, a composition may comprise two species of single oligonucleotides: ACTT-X and TCTT-X, where "X" is a sequence that is the same for both species of single oligonucleotides. It would be possible for the composition to comprise two copies of an oligonucleotide having the sequence ACTT-X, and one copy of an oligonucleotide having the sequence TCTT-X.

The oligonucleotide species of the compositions, methods and oligonucleotides comprise a barcode region and a uniform region as described herein. The barcode regions may differ between single oligonucleotide species, to provide diversity in a population of single oligonucleotide species, while the uniform regions remain the same. The barcode region may comprise a molecular index as described herein. The molecular index may be configured to minimize bias, for example by minimizing the G content such that no single oligonucleotide species in a population of single oligonucleotide species has a molecular index with a G content greater than 50%, and/ or in such a way that the sequence "GG" does not appear in the molecular index (for example, such that there are no two consecutive G's). The barcode region comprises a sample index. The sample index can be configured such that unique oligonucleotides from a given group can have the same sample index, but different molecular indexes. Thus, if multiple samples are analyzed, the sample index can indicate which sample each oligonucleotide corresponds to. In this way, after the single oligonucleotide species bind to the target, the single oligonucleotide species can be grouped and the sequences can be analyzed. In some embodiments, the index of the sample is 5' of the index of the molecule. In some embodiments, the index of the molecule is 5' of the index of the sample. Optionally, the single oligonucleotide species comprises an adapter. The adapter can be located 5' from the barcode region. In some embodiments, the adapter is configured to immobilize the single oligonucleotide species on a substrate.

Without being limited by any theory, it is contemplated that single oligonucleotide species configured in accordance with some embodiments herein can provide accurate analysis and sequencing results with reduced, minimal or no bias, for example by minimizing the G content in the barcode region or molecular barcode, and/or minimizing the G content near the uniform region. By way of example, a reduction in bias can be verified by a reduction in noise and/or an increase in sensitivity to detect the number of different molecules of a target nucleic acid in a sample (see, for example, FIGS. 6- 7). In some embodiments, the reduction in bias can be determined as a decrease in noise (see FIG. 6), for example, a smaller standard error in the number of target nucleic acid molecules detected in the sample. In some embodiments, the reduction in bias can be determined as an increase in sensitivity (see FIG. 7), for example, by detecting a greater number of different molecules of a target nucleic acid in a sample (e.g., they are "missed"). fewer target nucleic acid molecules). As such, in some embodiments, the compositions and methods allow for the quantification of target nucleic acid molecules in a sample with low noise, e.g., a relative standard error of less than 30%, e.g., less than 30%, 25%. , 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% or 0.01%, including intervals between two of the listed values. In some embodiments, the compositions and methods allow the quantification of target nucleic acid molecules in a sample with high sensitivity, e.g., a sensitivity (measured as a percentage of the actual number of different target nucleic acids in a sample that are detected). , for example, a sensitivity of at least 70%, 75%, 80%, 85%, 90%, 85%, 96%, 97%, 98%, 99% or 99.9%, including intervals between two any of the values listed. In some embodiments, the methods or compositions described herein reduce bias by increasing the sensitivity, reducing the relative standard error, or by increasing the sensitivity and reducing the standard error relative to compositions or methods comprising single oligonucleotide species in line with FIG. 3A.

FIG. 3A illustrates a conventional oligonucleotide design. It is noted that the molecular index sequence NNNNNNN (in which each N is A, G, C, or T, and in which any two of the N can be the same or different from each other) may comprise two or more consecutive Gs, and/or may have a G content of more than 50%. For example, a subset of the population of single oligonucleotide species according to FIG. 3A may be G-rich. Without being limited by any theory, it is contemplated that at least some unique oligonucleotide species in a population based on the configuration of FIG. 3A could be favored, leading to bias. Another example of a "reference" molecular index that may be subject to bias is the sequence 'BBBBBBV' (where B is C, G, or T, and where V is A, C, or G). In FIGS. 3B-3H illustrate examples of configurations for single oligonucleotide species according to some embodiments herein. As shown in FIGS. 3B-C, the addition of TTT or TTTTT after the molecular index according to some embodiments herein can reduce the G-richness throughout the region (the length of the non-G spacers can be variable, and can comprise any nucleotide or non-G nucleotides). In FIG. 3D, the sample index and molecule positions are swapped (such that the molecule index is 5' to the sample index), such that possible G-rich MIs in at least one subset of single oligonucleotide species would not be adjacent to potentially G-rich gene-specific target regions. In FIGS. 3E-F, the addition of TTT or TTTTT to the configuration illustrated in FIG. 3D can further minimize potentially G-rich regions. Without being limited by any theory, it is noted that sample barcodes may frequently have low G contents (e.g., to be "non-G-rich"). It is further noted that the length of the non-G spacers can be variable and can be any non-G nucleotide). In the configurations of FIGS. 3G-H, molecular barcodes comprise "HHHHHHHH" or "HNHNHNHN" (in which H is A, C or T, and in which any two H can be the same or different from each other, and in which any two N any can be the same or different from each other).

In some embodiments, each of a plurality of single oligonucleotide species (e.g., each single oligonucleotide species) in a composition or method has a length of at least 24 nucleotides, e.g., at least 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 55, 60, 65, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140 nucleotides in length, including intervals between two any of the values listed, for example 24 140, 24-135, 24-130, 24-125, 24-120, 24-115, 24-110, 24-105, 24-100, 24-95, 24-90 , 24-85, 24-80, 24-75, 24-70, 24 65, 24-60, 24-55, 24-50, 24-45, 24-40, 25-140, 25-135, 25- 130, 25-125, 25-120, 25-115, 25-110, 25-105, 25-100, 25 95, 25-90, 25-85, 25-80, 25-75, 25-70, 25 -65, 25-60, 25-55, 25-50, 25-45, 25-40, 27-140, 27-135, 27-130, 27-125, 27 120, 27-115, 27-110, 27-105, 27-100, 27-95, 27-90, 27-85, 27-80, 27-75, 27-70, 27-65, 27-60, 27-55, 27-50, 27- 45, 27-40, 30-140, 30-135, 30-130, 30-125, 30-120, 30-115, 30-110, 30-105, 30-100, 30-95, 30-90, 30-85, 30-80, 30-75, 30-70, 30-65, 30-60, 30-55, 30-50, 30-45, 30-40, 35-140, 35-135, 35- 130, 35-125, 35-120, 35-115, 35-110, 35-105, 35-100, 35-95, 35-90, 35-85, 35-80, 35-75, 35-70, 35-65, 35-60, 35-55, 35-50, 35-45, 35-40, 40-140, 40-135, 40-130, 40-125, 40-120, 40-115, 40- 110, 40-105, 40-100, 40-95, 40-90, 40-85, 40-80, 40-75, 40-70, 40-65, 40-60, 40-55, 40 50, or 40-45 nucleotides long. Optionally, different species of single oligonucleotides in a composition or method have different lengths from each other. Optionally, all single oligonucleotide species in a composition or method are of the same length as each other. Optionally, some species of single oligonucleotides in a composition or method have the same length as each other, while some species of single oligonucleotides have different lengths from each other.

In some embodiments, the uniform region comprises, consists of, or consists essentially of a 5' to 3' amplification sequence for a target nucleic acid, or class of target nucleic acids (this amplification sequence may also be referred to as a "target-specific" region. ). For example, if the target nucleic acids comprise mRNA, the uniform region may comprise oligo dT. For example, if the target nucleic acids comprise variable regions of a T cell receptor, the uniform region may comprise sequences flanking variable regions of a T cell receptor mRNA. In some embodiments, the uniform region comprises at least 10 nucleotides. that are complementary to the target nucleic acid, for example at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, or 30 nucleotides complementary to the target, including intervals between any two of the values listed, for example 10-30, 10-29, 10-28, 10 27, 10-26, 10-25, 10-24, 10 -23, 10-22, 10-21, 10-20, 11-30, 11-29, 11-28, 11-27, 11-26, 11-25, 11-24, 11-23, 11-22 , 11-21, 11-20, 12-30, 12-29, 12-28, 12-27, 12-26, 12-25, 12-24, 12-23, 12-22, 12-21, 12 -20, 15-30, 15-29, 15-28, 15 27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, or 20-21 nucleotides. In some embodiments, the uniform region comprises a target-specific region that hybridizes to a sequence flanking a sequence encoding a variable region of an immune cell receptor, for example a variable region of a T cell receptor, an immune cell receptor. B, or immunoglobulin, for example an antibody. It should be noted that, as B cell receptors comprise membrane-bound immunoglobulin, target regions specific for immunoglobulin variable region coding sequences are typically suitable for amplifying B cell receptors as well as secreted immunoglobulins (e.g., antibodies). Both options are indicated herein to clarify that amplification of membrane-bound immunoglobulins (B cell receptors) and also secreted immunoglobulins (antibodies) is contemplated. As used herein, it will be understood that when primers or uniform regions comprise target-specific regions that comprise "flanking sequences" (and variations of this root term, such as "flanking sequences") of immune cell receptor variable regions and/or immunoglobulins, the specific target regions will be understood to comprise at least one of (i) sequences that hybridize downstream (3') of the sequence encoding the variable region and, in particular, which hybridize to the coding sequence strand, and are therefore configured to produce a strand comprising the variable region coding sequence upon extension in the 5' to 3'direction; or (ii) sequences that hybridize upstream (5') of the sequence encoding the variable region and, in particular, hybridize to the strand complementary to that of the coding sequence and are therefore configured to produce a strand comprising the reverse complement of the coding sequence of the variable region after extension in the 5' to 3' direction. Therefore, a flanking sequence can be configured for amplification of the coding sequences of the variable region together with a suitable primer (for example, a flanking sequence on the other side of the variable region). It will be understood that a flanking sequence need not necessarily stop or start exactly where the coding sequence begins or stops and, therefore, it is permissible for there to be intermediate sequences between a hybridization site of a flanking sequence and the coding sequence of the region itself. variable. It will be understood that, although a flanking sequence generally hybridizes to a sequence external to the variable region coding sequence to amplify a wide variety of possible variable region coding sequences, in some embodiments, the "flanking sequence" of the variable region comprises also some sequence of the variable region itself, for example, if a subset of possible variable regions is of interest. However, a "flanking sequence," as used herein, does not require that a single sequence flank both sides of the variable region. Rather, it will be understood that when flanking sequences are mentioned in conjunction with compositions, methods and oligonucleotides of some embodiments herein, 5' and 3' sequences comprising primer pairs suitable for amplifying the variable region coding sequence are also expressly contemplated. .

A single oligonucleotide species comprises a barcode region as described herein and also comprises a uniform region comprising a target-specific region comprising a sequence flanking an immune cell receptor variable region coding sequence and/or or immunoglobulin. A second oligonucleotide primer may also be provided for the other side of the variable region coding sequence, to amplify the variable region sequence together with the target-specific region of the uniform region. The uniform region comprises a target-specific region, positioned 3' of the barcode region, and comprising a sequence that flanks an immunoglobulin variable region (and therefore flanks a B cell receptor variable region, as well as a corresponding antibody variable region), for example flanking the variable region of an immunoglobulin heavy chain locus, flanking the variable region of an immunoglobulin kappa (light chain) locus, or flanking the variable region of an immunoglobulin lambda locus (light chain). In some embodiments, the uniform region comprises a target-specific region, located 3' of the barcode region, and comprising a sequence flanking at least one of the variable regions of a T cell receptor alpha chain, a variable region of a T cell receptor beta chain, a variable region of a T cell receptor gamma chain, or a T cell receptor delta chain.

In some embodiments, a kit comprises a composition comprising single oligonucleotide species as described herein, wherein each of the single oligonucleotide species comprises a uniform sequence flanking an immune cell receptor variable region coding sequence. or immunoglobulin as described herein. The kit further comprises an oligonucleotide primer configured to hybridize to the opposite strand and on the other side of the variable region coding sequence as compared to the uniform region, and is therefore configured to amplify the variable region sequence together with the specific target of the uniform region. In some embodiments, the amplified sequence is at least 1 kb and comprises the variable coding sequence, for example at least 1 kb, 2 kb, 3 kb, 4 kb, or 5 kb, including intervals between any two of the listed values.

It should be noted that nucleic acids encoding variable regions of some immune cell receptors or immunoglobulins can be more than 1 kb in length. For example, T cell receptor variable region sequences may comprise a CDR3 coding sequence that ends more than 1 kb away from where the CDR1 coding sequence begins. Without being limited by theory, it is noted that some conventional and next generation sequencing approaches, for example sequencing by synthesis, are limited to short reads that are typically considerably less than 1 kb. Accordingly, it is contemplated that the methods, compositions and kits according to some embodiments herein may be useful for barcoding and analyzing nucleic acids that encode variable regions of immune cell receptors and/or immunoglobulins, which otherwise Otherwise they would not be amenable to single read sequencing of less than 1 kb. Accordingly, in some embodiments, the single oligonucleotide species comprises a sequence that flanks an immune cell receptor or immunoglobulin variable region coding sequence, is configured to amplify a sequence of at least 1 kb, and comprises the coding sequence of variable region, for example at least 1 kb, 2 kb, 3 kb, 4 kb or 5 kb, including intervals between any two of the listed values.

Regions with barcodes

According to the compositions, methods and oligonucleotides of some embodiments herein, a barcode region comprises a nucleic acid sequence that is useful for identifying a nucleic acid, for example a target nucleic acid from a sample, or an amplicon or reverse transcript derived from a single target nucleic acid from a sample. For example, two mRNA transcripts from a sample can be reverse transcribed and barcoded such that the nucleic acids corresponding to the first mRNA include a first barcode, and the nucleic acids corresponding to the second mRNA include a second code. of bars. After sequencing (or other analysis), information about individual mRNAs in the sample, for example copy number, can be obtained even after amplification. However, if a large population of mRNA is stochastically labeled and some barcodes are represented more favorably (e.g. due to stability, amplification efficiency, etc.), bias may occur, skewing the ability to quantify the nucleic acids from a sample. Therefore, according to some embodiments herein, each unique oligonucleotide species in a population may comprise a unique barcode region. The greater the barcode diversity, the greater the diversity of single oligonucleotide species, and the greater the probability that a particular barcode sequence will be associated with a single target nucleic acid in a sample. In some embodiments, methods or compositions comprising single oligonucleotide species comprising molecular barcodes as described herein reduce bias by increasing sensitivity, reducing relative standard error, or increasing sensitivity and reducing standard error. As used herein, a “molecule barcode” may also be referred to as a “molecular barcode,” “molecular index (MI),” or unique molecular identifier (IMU). Herein, a "sample barcode" may also be referred to as a "sample index (SI)".

A barcode region may comprise a molecular barcode. The molecular barcode may comprise a unique sequence, such that when multiple nucleic acids of the sample (which may be the same and/or different from each other) are associated one by one with molecular barcodes, the different nucleic acids of The sample can be differentiated from each other by molecular barcodes. As such, even if a sample comprises two nucleic acids with the same sequence, each of these two nucleic acids can be labeled with a different molecular barcode, such that nucleic acids in the population can be quantified, even after amplification. The molecular barcode may comprise a nucleic acid sequence of at least 5 nucleotides, for example at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides, including intervals between any two of the values listed, for example 5-50, 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6- 50, 6-45, 6-40, 6-35, 6 30, 6-25, 6-20, 6-15, 6-14, 6-13, 6-12, 6-11,6-10, 6 -9, 6-8, 6-7, 7-50, 7-45, 7-40, 7-35, 7-30, 7-25, 7-20, 7-15, 7-14, 7 13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-50, 8-45, 8-40, 8-35, 8-30, 8-25, 8-20, 8- 15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-50, 9 45, 9-40, 9-35, 9-30, 9-25, 9 -20, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25 , 10-20, 10-15, 10-14, 10-13, 10-12, or 10-11 nucleotides. In some embodiments, the nucleic acid sequence of the molecular barcode comprises a unique sequence, for example, such that each unique oligonucleotide species in a composition comprises a different molecular barcode. In some embodiments, two or more unique oligonucleotide species may comprise the same molecular barcode, but still differ from each other. For example, if the unique oligonucleotide species include sample barcodes, each unique oligonucleotide species with a particular sample barcode may comprise a different molecular barcode. The claimed composition comprising unique oligonucleotide species comprises a molecular barcode diversity of at least 1000 different molecular barcodes, and therefore at least 1000 unique oligonucleotide species. In some embodiments, a composition comprising unique oligonucleotide species comprises a molecular barcode diversity of at least 6,500 different molecular barcodes, and therefore at least 6,500 unique oligonucleotide species. In some embodiments, a composition comprising unique oligonucleotide species comprises a molecular barcode diversity of at least 65,000 different molecular barcodes, and therefore at least 65,000 unique oligonucleotide species.

Without being limited by any theory, it is contemplated that a molecular barcode comprising a low G content (e.g., 50% G or less, e.g., less than 50% G, 45% G, 40% G, 35% of G, 30% of G, 25% of G, 20% of G, 15% of G, 12.5% of G, 10% of G, 7.5% of G, 5% of G , or 2.5% G) can minimize the bias for a composition or group of single oligonucleotide species that are used to barcode a population of nucleic acids (e.g., minimize the bias that would preferentially amplify barcodes which comprise a higher G content). It is noted that conventional barcode coding approaches typically show a bias in favor of higher G content. For example, FIGS. 4A, 4B, and 4C illustrate samples of nucleotide usage in conventional molecular barcodes of compositions comprising numerous unique molecular barcodes for ES32, TRAC (FIG. 4A), ES32 TRBC (FIG. 4B), and ES32 OligodT ( FIG. 4C). That is, if anything, conventional molecular barcodes and barcode regions, designed without respecting certain orientations provided herein, may comprise a higher G content than would be expected by random chance. In some embodiments, methods or compositions comprising single oligonucleotide species comprising molecular barcodes as described herein reduce bias by increasing sensitivity, reducing relative standard error, or increasing sensitivity and reducing standard error.

In some embodiments, all molecular barcodes of a composition or composition used in the methods described herein comprise single oligonucleotide species that collectively have a G content of less than 12.5% G, 10% G, 7 .5% of G, 5% of G, or 2.5% of G, or 0% of G, including intervals between any two of the values listed, for example 2.5-10% of G, 2.5- 7.5% G, 2.5-5% G, 5-10% G, 5-7.5% G, 7.5-10% G, 10-12.5% G. By "all molecular barcodes in a single oligonucleotide species composition collectively have a G content of...", it is meant that if the total G content among all molecular barcodes in the entire composition were calculated (e.g., a population of at least 1000, 2000, 5000, 6500, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, or 65,000 unique oligonucleotide species), this total G content of the total sum of the codes of bars would fall below the listed values or within the listed ranges. Although it would still be possible for an individual single oligonucleotide species to have a molecular barcode with a G content above the indicated value or outside the indicated range, the collective nucleotide content of the single oligonucleotide species in the composition would be below of the indicated value or within the indicated range. In some embodiments, all molecular barcodes in a composition comprising at least 1000 unique oligonucleotide species collectively have a G content of less than 12.5% G, 10% G, 7.5% G , 5% of G, or 2.5% of G, or 0% of G, including intervals between any two of the indicated values. In some embodiments, all molecular barcodes in a composition comprising at least 6500 unique oligonucleotide species collectively have a G content of less than 12.5% G, 10% G, 7.5% G, 5% of G, or 2.5% of G, or 0% of G, including intervals between any two of the values listed. In some embodiments, all molecular barcodes in a composition comprising at least 65,000 unique oligonucleotide species collectively have a G content of less than 12.5% G, 10% G, 7.5% G , 5% of G, or 2.5% of G, or 0% of G, including intervals between any two of the values listed.

In some embodiments, for a composition comprising single oligonucleotide species (or such composition used in a method), the composition consists of or essentially consists of single oligonucleotide species each comprising a molecular barcode G content of 50 % G or less, for example, less than 50% G, 45% G, 40% G, 35% G, 30% G, 25% G, 20% G, 15% G , 12.5% of G, 10% of G, 7.5% of G, 5% of G, or 2.5% of G, or 0% of G, including intervals between any two of the values listed, for example 2 .5-50% of G, 2.5-45% of G, 2.5-40% of G, 2.5-35% of G, 2.5-30% of G, 2.5-25% of G, 2.5-20% of G, 2.5-15% of G, 2.5-10% of G, 2.5-7.5% of G, 2.5-5% of G, 5- 50% of G, 5-45% of G, 5-40% of G, 5-35% of G, 5-30% of G, 5-25% of G, 5-20% of G, 5-15 % G, 5-10% G, 5-7.5% G, 7.5-50% G, 7.5-45% G, 7.5-40% G, 7.5 -35% of G, 7.5-30% of G, 7.5-25% of G, 7.5-20% of G, 7.5-15% of G, 7.5-10% of G, 10 -50% of G, 10-45% of G, 10-40% of G, 10-35% of G, 10-30% of G, 10-25% of G, 10-20% of G, 10- 15% G, 10-12.5% G, 12.5-50% G, 12.5-45% G, 12.5-40% G, 12.5-35% G, 12, 5-30% G, 12.5-25% G, 12.5-20% G, 12.5-15% G, 15-50% G, 15-45% G, 15- 40% G, 15-35% G, 15-30% G, 15-25% G, 15-20% G, 20-50% G, 20-45% G, 20-40 % G, 20-35% G, 20-30% G, or 20-25% G. By "the composition consists of, or consists essentially of, unique oligonucleotide species each having a code content of molecular barcode G of less than...", it is understood that each or essentially each of the unique oligonucleotide species in a composition, population or group has a molecular barcode G content less than the indicated value, or outside the indicated range. That is, for a composition, population, or group "consisting essentially of" species of single oligonucleotides each having the indicated G content, it would be possible for an analytically insignificant portion of the single oligonucleotides in the composition to have molecular barcodes with a G content above the indicated value or outside the listed range. For example, the analytically insignificant part of the unique oligonucleotides may have, or may have no more than 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or less of the unique oligonucleotide in a composition. In some embodiments, less than 1% of the unique oligonucleotide species in a composition comprising at least 1000 unique oligonucleotides comprise a molecular barcode having a G content greater than 50%, for example less than 1%, 0 .9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0 .01%, 0.001%, or 0.0001% of the single oligonucleotide species have a G content greater than 50%. In some embodiments, less than 1% of the unique oligonucleotide species in a composition comprising at least 6500 unique oligonucleotides comprise a molecular barcode having a G content greater than 50%, for example less than 1%, 0 .9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0 .01%, 0.001%, or 0.0001% of the single oligonucleotide species have a G content greater than 50%. In some embodiments, less than 1% of the unique oligonucleotide species in a composition comprising at least 65,000 unique oligonucleotides comprise a molecular barcode having a G content greater than 50%, for example less than 1%, 0 .9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0 .01%, 0.001%, or 0.0001% of the single oligonucleotide species have a G content greater than 50%. In some embodiments, less than 1% of the unique oligonucleotide species in a composition comprising at least 1000 unique oligonucleotides comprise a molecular barcode having a G content greater than 25%, for example less than 1%, 0 .9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0 .01%, 0.001%, or 0.0001% of the single oligonucleotide species have a G content greater than 25%. In some embodiments, less than 1% of the unique oligonucleotide species in a composition of at least 6500 unique oligonucleotides comprise a molecular barcode having a G content greater than 25%, for example less than 1%, 0, 9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.01%, 0.001%, or 0.0001% of the single oligonucleotide species have a G content greater than 25%. In some embodiments, less than 1% of the unique oligonucleotide species in a composition, population or group of at least 65,000 unique oligonucleotides comprise a molecular barcode that has a G content greater than 25%, for example less than 1 %, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05 %, 0.01%, 0.001%, or 0.0001% of the single oligonucleotide species have a G content greater than 25%. Optionally, none of the molecular barcodes of the single oligonucleotide species of the composition collectively have a G content greater than 50% G, for example, all of the molecular barcodes of the single oligonucleotide species have a G content. of less than 50% of G, 45% of G, 40% of G, 35% of G, 30% of G, 25% of G, 20% of G, 15% of G, 12.5% of G, 10% of G, 7.5% of G, 5% of G, or 2.5% of G, or 0% of G, including intervals between any two of the values listed.

In some embodiments, the composition as described herein (or said composition as used in a method) consists of or consists essentially of single oligonucleotide species each comprising a G barcode region with a content of less than 50% as described herein. In some embodiments, for a composition comprising single oligonucleotide species, the composition consists of or essentially consists of single oligonucleotide species each comprising a barcode region G content of 50% G or less, e.g. less than 50% of G, 45% of G, 40% of G, 35% of G, 30% of G, 25% of G, 20% of G, 15% of G, 12.5% of G, 10% of G, 7.5% of G, 5% of G, or 2.5% of G, or 0% of G, including intervals between any two of the values listed, for example 2.5-50% of G, 2 .5-45% of G, 2.5-40% of G, 2.5-35% of G, 2.5-30% of G, 2.5-25% of G, 2.5-20% of G , 2.5-15% G, 2.5-10% G, 2.5-7.5% G, 2.5-5% G, 5-50% G, 5-45% of G, 5-40% of G, 5-35% of G, 5-30% of G, 5-25% of G, 5-20% of G, 5-15% of G, 5-10% of G, 5-7.5% G, 7.5-50% G, 7.5-45% G, 7.5-40% G, 7.5-35% G, 7.5-30 % G, 7.5-25% G, 7.5-20% G, 7.5-15% G, 7.5-10% G, 10-50% G, 10-45% G, 10-40% of G, 10-35% of G, 10-30% of G, 10-25% of G, 10-20% of G, 10-15% of G, 10-12.5% of G, 12.5-50% of G, 12.5-45% of G, 12.5-40% of G, 12.5-35% of G, 12.5-30% of G, 12, 5-25% G, 12.5-20% G, 12.5-15% G, 15-50% G, 15-45% G, 15-40% G, 15-35% G , 15-30% of G, 15-25% of G, 15-20% of G, 20-50% of G, 20-45% of G, 20-40% of G, 20-35% of G, 20-30% G, or 20-25% G. For "the composition consists of or consists essentially of single oligonucleotide species each having a barcode G region content of less than....", It is understood that each or essentially each of the single oligonucleotide species in a composition, population or group has a barcode region G content less than the indicated value, or outside the indicated range. That is, for a composition, population, or group "consisting essentially of" species of single oligonucleotides each having the indicated G content, it would be possible for an analytically insignificant portion of the single oligonucleotides in the composition to have barcode regions with a G content above the indicated value or outside the mentioned range. In some embodiments, less than 1% of the unique oligonucleotide species in a composition comprising at least 1000 unique oligonucleotides comprise a barcode region having a G content of more than 50%, for example less than 1%. , 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05% , 0.01%, 0.001%, or 0.0001% of the single oligonucleotide species have a G content of more than 50%, including intervals between any two of the values listed. In some embodiments, less than 1% of the unique oligonucleotide species in a composition comprising at least 6500 unique oligonucleotides comprise a barcode region having a G content of more than 50%, for example less than 1%. , 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05% , 0.01%, 0.001%, or 0.0001% of the single oligonucleotide species have a G content of more than 50%, including intervals between any two of the values listed. In some embodiments, less than 1% of the unique oligonucleotide species in a composition comprising at least 65,000 unique oligonucleotides comprise a barcode region having a G content of more than 50%, for example less than 1%. , 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05% , 0.01%, 0.001%, or 0.0001% of the single oligonucleotide species have a G content of more than 50%, including intervals between any two of the values listed. In some embodiments, less than 1% of the unique oligonucleotide species in a composition comprising at least 1000 unique oligonucleotides comprise a barcode region having a G content of more than 25%, for example less than 1%. , 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05% , 0.01%, 0.001%, or 0.0001% of the single oligonucleotide species have a G content of more than 25%, including intervals between any two of the values listed. In some embodiments, less than 1% of the unique oligonucleotide species in a composition of at least 6500 unique oligonucleotides comprise a barcode region having a G content of more than 25%, for example less than 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.01%, 0.001%, or 0.0001% of the single oligonucleotide species have a G content of more than 50%, including intervals between any two of the values listed. In some embodiments, less than 1% of the unique oligonucleotide species in a composition, population or pool of at least 65,000 unique oligonucleotides comprise a barcode region having a G content of more than 25%, e.g. 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0 .05%, 0.01%, 0.001%, or 0.0001% of the single oligonucleotide species have a G content of more than 25%, including intervals between any two of the values listed. Optionally, none of the barcode regions of the single oligonucleotide species of the composition collectively have a G content of more than 50% G, for example, all barcode regions of the single oligonucleotide species have a G content of less than 50% G, 45% G, 40% G, 35% G, 30% G, 25% G, 20% G, 15% G, 12.5 %G, 10%G, 7.5% of G, 5% of G, or 2.5% of G, or 0% of G, including intervals between any two of the values listed.

In some embodiments, the composition (or said composition as used in a method) consists of or consists essentially of single oligonucleotide species each having a molecular barcode comprising the sequence of at least three repeats of the "HN" doublet. (in which each "H" is any of A, C, or T, and in which "N" is any of A, G, C, or T), for example at least 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repetitions, including intervals between any two of the listed values. Examples of multiple repeats of the "HN" doublet include HN, HNHN, HNHNHN, and HNHNHNHN. It should be noted that although the "HN" formula describes restrictions on base content, not all H's or all N's have to be the same or different. For example, if the molecular barcodes of single oligonucleotide species in a composition comprised HNHNHN, one molecular barcode may comprise the sequence ACTGCA, while another molecular barcode may comprise the sequence TAACTA, while another barcode molecular could comprise the sequence AGACAc. It should be noted that any number of repeats of the "HN" doublet would have a G content of no more than 50%. In some embodiments, at least 95% of the unique oligonucleotide species of a composition comprising at least 1000 unique oligonucleotide species comprise molecular barcodes comprising at least three repeats of the "HN" doublet, for example by at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repetitions, including intervals between any two of the values listed. In some embodiments, at least 99% of the unique oligonucleotide species of a composition comprising at least 1000 unique oligonucleotide species comprise molecular barcodes comprising at least three repeats of the "HN" doublet, for example by at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repetitions, including intervals between any two of the values listed. In some embodiments, at least 99.9% of the unique oligonucleotide species of a composition comprising at least 1000 unique oligonucleotide species comprise molecular barcodes comprising at least three repeats of the "HN" doublet, e.g. example at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repetitions, including intervals between two of the listed values . In some embodiments, at least 95% of the unique oligonucleotide species of a composition comprising at least 6500 unique oligonucleotide species comprise molecular barcodes comprising at least three repeats of the "HN" doublet, for example by at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repetitions, including intervals between two of the listed values. In some embodiments, at least 99% of the unique oligonucleotide species of a composition comprising at least 6500 unique oligonucleotide species comprise molecular barcodes comprising at least three repeats of the "HN" doublet, for example by at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repetitions, including intervals between two of the listed values. In some embodiments, at least 99.9% of the unique oligonucleotide species of a composition comprising at least 6500 unique oligonucleotide species comprise molecular barcodes comprising at least three repeats of the "HN" doublet, e.g. example at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repetitions, including intervals between two of the listed values . In some embodiments, at least 95% of the unique oligonucleotide species of a composition comprising at least 65,000 unique oligonucleotide species comprise molecular barcodes comprising at least three repeats of the "HN" doublet, for example by at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repetitions, including intervals between two of the listed values. In some embodiments, at least 99% of the unique oligonucleotide species of a composition comprising at least 65,000 unique oligonucleotide species comprise molecular barcodes comprising at least three repeats of the "HN" doublet, for example by at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repetitions, including intervals between any two of the values listed. In some embodiments, at least 99.9% of the unique oligonucleotide species of a composition comprising at least 65,000 unique oligonucleotide species comprise molecular barcodes comprising at least three repeats of the "HN" doublet, e.g. example at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repetitions, including intervals between two of the listed values . In some embodiments, the composition consists of or essentially consists of at least 1000, 6500, or 65,000 species of unique oligonucleotides each having a molecular barcode comprising the sequence HNHNHN. In some embodiments, the composition consists of or essentially consists of at least 1000, 6500 or 65,000 species of unique oligonucleotides each having a molecular barcode comprising the sequence HNHNHN. In some embodiments, at least 95%, 99%, or 99.9% of the barcode regions of the composition described herein comprise at least three repeats of the "HN" doublet described herein. Without being limited by any theory, it is noted that having a relatively large number of nucleotide sequences available for molecular barcoding can be useful when barcoding a population of target nucleic acids from a sample, for example to increase the barcode diversity within a given sequence length along with the probability that each target nucleic acid is uniquely labeled, while minimizing the size of the oligonucleotide species. It is noted that limiting the G content of molecular barcodes and/or barcode regions can limit the diversity of these barcodes and barcode regions by reducing the number of available nucleotides from which they can be barcodes can be constructed (and the number of different sequences available per nucleic acid length). As such, having some Gs in the molecular barcodes or barcode regions according to various embodiments herein may be useful to increase diversity, while limiting G content may be useful to minimize bias. It is indicated, and has been observed (see Example 2 and FIGS. 6-7) that sequences comprising repeated "HN" doublets can produce low bias, while providing a compromise between bias reduction and maintenance of a relatively large number of available nucleotide sequences, such that relatively high diversity can be obtained in a relatively short sequence, while minimizing bias. In some embodiments, methods or compositions comprising single oligonucleotide species comprising molecular barcodes comprising repeated "HN" doublets as described herein reduce bias by increasing sensitivity, reducing relative standard error, or increasing sensitivity. and reducing the standard error.

In some embodiments, the composition (or such composition as used in a method) comprises, consists of, or consists essentially of single oligonucleotide species each comprising a molecular barcode comprising at least six consecutive "H"s ( in which each "H" is any of A, C, or T), for example at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , or 20 consecutive H, including intervals between any two of the values listed. It should be noted that although the "H" formula describes restrictions on base content, not all H's have to be the same (or different). For example, if the molecular barcodes of single oligonucleotide species in a population each comprised the sequence HHHH, a molecular barcode of one single oligonucleotide species could comprise ACTA, a molecular barcode of another single oligonucleotide species could comprise Tt AC, and a molecular barcode of another single oligonucleotide species could comprise ACAT. In some embodiments, a composition comprises, consists of, or consists essentially of at least 1000 unique oligonucleotide species, at least 95% of which comprise a molecular barcode comprising at least six consecutive H's, e.g. at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive hours, including intervals between any two of the values listed. In some embodiments, a composition comprises, consists of, or essentially consists of at least 1000 unique oligonucleotide species, at least 99% of which comprise a molecular barcode comprising at least six consecutive H's, e.g. at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive hours, including intervals between any two of the values listed. In some embodiments, a composition comprises, consists of, or consists essentially of at least 1000 unique oligonucleotide species, at least 99.9% of which comprise a molecular barcode comprising at least six consecutive H, for example at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive H, including intervals between any two of the values listed. In some embodiments, a composition comprises, consists of, or consists essentially of at least 6500 unique oligonucleotide species, at least 95% of which comprise a molecular barcode comprising at least six consecutive H, for example by at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive hours, including intervals between any two of the values listed. In some embodiments, a composition comprises, consists of, or consists essentially of at least 6500 unique oligonucleotide species, at least 99% of which comprise a molecular barcode comprising at least six consecutive H's, e.g. at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive hours, including intervals between any two of the values listed. In some embodiments, a composition comprises, consists of, or consists essentially of at least 6500 unique oligonucleotide species, at least 99.9% of which comprise a molecular barcode comprising at least six consecutive H, for example at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive H, including intervals between any two of the values listed. In some embodiments, a composition comprises, consists of, or consists essentially of at least 65,000 unique oligonucleotide species, at least 95% of which comprise a molecular barcode comprising at least six consecutive H's, e.g. at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive hours, including intervals between any two of the values listed. In some embodiments, a composition comprises, consists of, or consists essentially of at least 65,000 unique oligonucleotide species, at least 99% of which comprise a molecular barcode comprising at least six consecutive H's, e.g. at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive hours, including intervals between any two of the values listed. In some embodiments, a composition comprises, consists of, or consists essentially of at least 65,000 unique oligonucleotide species, at least 99.9% of which comprise a molecular barcode comprising at least six consecutive Hs, for example at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive H, including intervals between any two of the values listed. In some embodiments, at least 95% of the molecular barcodes of the unique oligonucleotide species comprise a sequence that sums to at least 6 alternating H and N, for example at least 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 alternating H and N, including intervals between any two of the listed values. In some embodiments, at least 95% of the molecular barcodes of the single oligonucleotide species comprise the sequence HNHNHNHN, where each "H" is any one of A, C or T, and where each "N" is any one of A, G, C or T. In some embodiments, each molecular barcode of the single oligonucleotide species comprises the sequence HNHNHNHN, where each "H" is any one of A, C or T, and in where each "N" is any one of A, G, C or T. In some embodiments, each molecular barcode of the unique oligonucleotide species comprises the sequence HHHHHHHH, where each "H" is any one of A, C or T.

In some embodiments, at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or more) of the unique oligonucleotide species of the composition as described herein (or such composition as used in a method) comprise barcode regions comprising at least 6 consecutive H, for example at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive H, including intervals between any two of the values listed as described herein. In some embodiments, at least 99% of the unique oligonucleotide species of the composition as described herein comprise barcode regions comprising at least 6 consecutive H, e.g. at least 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive H, including intervals between any two of the values listed as described herein. In some embodiments, at least 99.9% of the unique oligonucleotide species of the composition as described herein comprise barcode regions comprising at least 6 consecutive H, e.g. at least 6.7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive H, including intervals between any two of the values listed as described herein.

In some embodiments, the sample barcode of each single oligonucleotide species has a G content of 50% or less, for example 50% or less, 40% or less, 25% or less, 20% or less, 12.5% or less, 10% or less, or 5% or less, including intervals between any two of the values listed.

In some embodiments, the barcode region of each single oligonucleotide species has a G content of 50% or less, for example 50% or less, 40% or less, 25% or less, 20% or less, 12 .5% or less, 10% or less, or 5% or less, including intervals between any two of the values listed.

In some embodiments, the molecular barcodes of the single oligonucleotide species collectively have a G content of less than 12.5%, for example less than 12.5%, less than 10%, less than 7, 5%, less than 5%, less than 2.5%, or less than 1%, including intervals between any two of the listed values.

In some embodiments, the barcode regions of the single oligonucleotide species collectively have a G content of less than 12.5%, for example less than 12.5%, less than 10%, less than 7 .5%, less than 5%, less than 2.5%, or less than 1%, including intervals between any two of the listed values.

In some embodiments, for at least 95% of the unique oligonucleotide species, any G in the molecular barcode is not adjacent to another G. In some embodiments, for at least 99% of the unique oligonucleotide species , any G in the molecular barcode is not adjacent to another G. In some embodiments, for all or substantially all single oligonucleotide species, any G in the molecular barcode is not adjacent to another G.

Each barcode region comprises a sample barcode. According to the compositions, methods and oligonucleotides of some embodiments herein, each unique oligonucleotide species in a pool may comprise the same sample barcode, but there may be two or more pools that are each associated with different barcodes. sample bars. As such, all or essentially all of the unique oligonucleotide species in group No. 1 may comprise the sample barcode No. 1, and all or essentially all of the unique oligonucleotide species in group No. 2 may comprise the code sample barcode No. 2. The nucleic acids of a first sample may comprise the same sample barcode. Nucleic acids from a first sample can be associated with the unique oligonucleotide species in group No. 1, and nucleic acids from a second sample can be associated with the unique oligonucleotide species in group No. 2, for example by hybridization and amplification. As such, all or essentially all amplified nucleic acids corresponding to the first sample will comprise the sample barcode #1 (but may comprise different molecular barcodes), and all amplified nucleic acids corresponding to the second sample will comprise the sample barcode No. 2. In some embodiments, there are at least 24, 48, 96 or 192 groups.

The sample barcode may comprise a nucleic acid sequence of at least 3 nucleotides, for example of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39 , 40, 41,42, 43, 44, 45, 46, 47, 4849, 50 nucleotides, including intervals between any two of the values listed, for example 3-50, 3-45, 3-40, 3-35, 3-30, 3-25, 3-20, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3- 6, 3-5, 3-4, 4-50, 4-45, 4-40, 4-35, 4-30, 4-25, 4-20, 4-15, 4-14, 4-13, 4 12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-50, 5-45, 5-40, 5-35, 5-30 , 5-25, 5-20, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6 -50, 6-45, 6-40, 6-35, 6-30, 6-25, 6-20, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10 , 6-9, 6-8, 6-7, 7-50, 7-45, 7 40, 7-35, 7-30, 7-25, 7-20, 7-15, 7-14, 7- 13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-50, 8-45, 8-40, 8-35, 8-30, 8-25, 8-20, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-50, 9-45, 9-40, 9-35, 9-30, 9- 25, 9-20, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 10-14, 10-13, 10-12, or 10-11 nucleotides. In some embodiments, the sample barcode nucleic acid sequence comprises a unique sequence, e.g. such that each unique oligonucleotide species in a population comprises a different molecular barcode.

Without being limited by any theory, it is contemplated that a sample barcode comprising a low G content (e.g., less than 50% G, e.g., less than 45% G, 40% G, 35 % of G, 30% of G, 25% of G, 20% of G, 15% of G, 12.5% of G, 10% of G, 7.5% of G, 5% of G or 2, 5% G) can be placed 3' of a sample barcode comprising a relatively higher G content and 5' of a uniform region (e.g., a target-specific sequence or oligo dT sequence), to minimize the bias separating the relatively G-rich sample barcode from the uniform region. In some embodiments, the barcode region comprises a sample barcode with a G content of 50% or less, for example, less than 50% G, 45% G, 40% G, 35% G, 30% of G, 25% of G, 20% of G, 15% of G, 12.5% of G, 10% of G, 7.5% of G, 5% of G, or 2.5% of G, or 0% of G, including intervals between any two of the values listed, for example 2.5-50% of G, 2.5-45% of G, 2.5-40% of G, 2, 5-35% G, 2.5-30% G, 2.5-25% G, 2.5-20% G, 2.5-15% G, 2.5-10% G, 2.5-7.5% G, 2.5-5% G, 5-50% G, 5-45% G, 5-40% G, 5-35% G, 5- 30% G, 5-25% G, 5-20% G, 5-15% G, 5-10% G, 5-7.5% G, 7.5-50% G, 7 .5-45% of G, 7.5-40% of G, 7.5-35% of G, 7.5-30% of G, 7.5-25% of G, 7.5-20% of G, 7.5-15% of G, 7.5-10% of G, 10-50% of G, 10-45% of G, 10-40% of G, 10-35% of G, 10 -30% of G, 10-25% of G, 10-20% of G, 10-15% of G, 10-12.5% of G, 12.5-50% of G, 12.5-45% of G, 12.5-40% of G, 12.5-35% of G, 12.5-30% of G, 12.5-25% of G, 12.5- 20% of G, 12.5-15 % G, 15-50% G, 15-45% G, 15-40% G, 15-35% G, 15-30% G, 15-25% G, 15-20% of G, 20-50% of G, 20-45% of G, 20-40% of G, 20-35% of G, 20-30% of G, or 20-25% of G.

In some embodiments, for a composition comprising single oligonucleotide species (or such composition as used in a method), at least 95% of the sample barcodes of the single oligonucleotides of the composition each have less than 50% G content, for example, less than 45% G, 40% G, 35% G, 30% G, 25% G, 20% G, 15% G, 12.5% G, 10% of G, 7.5% of G, 5% of G, or 2.5% of G, or 0% of G, including intervals between any two of the values listed, for example, 2.5- 50% of G, 2 .5-45% of G, 2.5-40% of G, 2.5-35% of G, 2.5-30% of G, 2.5-25% of G, 2.5-20% of G, 2.5-15% of G, 2.5-10% of G, 2.5-7.5% of G, 2.5-5% of G, 5-50% of G, 5- 45% of G, 5-40% of G, 5-35% of G, 5-30% of G, 5-25% of G, 5-20% of G, 5-15% of G, 5-10 % G, 5-7.5% G, 7.5-50% G, 7.5-45% G, 7.5-40% G, 7.5-35% G, 7 .5-30% of G, 7.5-25% of G, 7.5-20% of G, 7.5-15% of G, 7.5-10% of G, 10-50% of G , 10-45% of G, 10-40% of G, 10-35% of G, 10-30% of G, 10-25% of G, 10-20% of G, 10-15% of G, 10-12.5% G, 12.5-50% G, 12.5-45% G, 12.5-40% G, 12.5-35% G, 12.5-30 % G, 12.5-25% G, 12.5-20% G, 12.5-15% G, 15-50% G, 15-45% G, 15-40% G, 15-35% of G, 15-30% of G, 15-25% of G, 15-20% of G, 20-50% of G, 20-45% of G, 20-40% of G , 20-35% of G, 20-30% of G or 20-25% of G.

In some embodiments, for a composition comprising single oligonucleotide species (or such composition as used in a method), at least 99% of the sample barcodes of the unique oligonucleotides of the composition each have a content of 50% G or less, for example, less than 50% G, 45% G, 40% G, 35% G, 30% G, 25% G, 20% G, 15% G, 12.5% of G, 10% of G, 7.5% of G, 5% of G, or 2.5% of G, or 0% of G, including intervals between any two of the listed values, e.g. 2.5-50% G, 2.5-45% G, 2.5-40% G, 2.5-35% G, 2.5-30% G, 2.5-25% G, 2.5-20% of G, 2.5- 15% of G, 2.5-10% of G, 2.5-7.5% of G, 2.5-5% of G, 5-50 % G, 5-45% G, 5-40% G, 5-35% G, 5-30% G, 5-25% G, 5-20% G, 5-15% of G, 5-10% of G, 5-7.5% of G, 7.5-50% of G, 7.5-45% of G, 7.5-40% of G, 7.5-35% of G, 7.5-30% of G, 7.5-25% of G, 7.5-20% of G, 7.5-15% of G, 7.5-10% of G, 10-50% of G, 10-45% of G, 10-40% of G, 10-35% of G, 10-30% of G, 10-25% of G, 10-20% of G, 10-15% of G, 10-12.5% of G, 12.5- 50% of G, 12.5-45% of G, 12.5-40% of G, 12.5-35% of G, 12.5-30 % G, 12.5-25% G, 12.5-20% G, 12.5- 15% G, 15-50% G, 15-45% G, 15-40% G, 15- 35% G, 15-30% G, 15-25% G, 15-20% G, 20-50% G, 20-45% G, 20-40% G, 20-35 % G, 20-30% G, or 20-25% G. That is, within the population of single oligonucleotides, less than 1% of the barcodes in the sample have a G content of more than fifty%. In some embodiments, the single oligonucleotide species of the composition consist of or consist essentially of single oligonucleotide species each having sample barcodes with a G content of less than 50%, as described herein.

In some embodiments, a barcode region is located 5' of the uniform region without any intervening sequence between the barcode region and the uniform region. In some embodiments, the barcode region is located 5' of a spacer, which is located 5' of the uniform region, such that there is a spacer between the barcode region and the uniform region. Optionally, the spacer has a low G content (e.g., 50% G or less, e.g., less than 50% G, 45% G, 40% G, 35% G, 30% G , 25% of G, 20% of G, 15% of G, 12.5% of G, 10% of G, 7.5% of G, 5% of G, or 2.5% of G). The spacer may be at least 1 nucleotide long, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 25, 30, 35, 40, 45, or 50 nucleotides, including intervals between any two of the values listed, for example 1-50, 1-45, 1-35, 1-30, 1-25 , 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2 45, 2-35, 2-30, 2-25, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-50, 3-45, 3-35, 3-30, 3-25, 3-20, 3-15, 3-10, 3 9, 3-8, 3-7, 3-6 , 3-5, 3-4, 4-50, 4-45, 4-35, 4-30, 4-25, 4-20, 4-15, 4-10, 4-9, 4-8, 4 -7, 4-6, 4-5, 5-50, 5-45, 5-35, 5-30, 5-25, 5-20, 5-15, 5-10, 5-9, 5-8 , 5-7, 5-6, 6-50, 6-45, 6-35, 6-30, 6-25, 6-20, 6-15, 6-10, 6-9, 6-8, 6 -7, 7-50, 7-45, 7-35, 7-30, 7-25, 7-20, 7-15, 7-10, 7-9, 7-8, 8-50, 8-45, 8-35, 8-30, 8-25, 8- 20, 8-15, 8-10, 8-9, 9-50, 9-45, 9-35, 9-30, 9-25, 9-20, 9-15, 9-10, 10-50, 10-45, 10-35, 10-30, 10-25, 10-20, or 10-15 nucleotides. In some embodiments, the spacer may comprise at least two consecutive non-G nucleotides (denoted "H", where "H" is A, C or T), for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive hours, including intervals between any two of the listed values, for example 2-20, 2- 15, 2-10, 2-9, 2-8, 2-7, 2 6, 2-5, 2-4, 2-3, 3-20, 3-15, 3-10, 3-9, 3 -8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-15, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5 , 5-20, 5-15, 5-10, 5-9, 5-8, 5-7, 5-6, 6-20, 6-15, 6-10, 6-9, 6-8, or 6-7. In some embodiments, the spacer nucleotides are Ts. For example, the spacer may comprise a poly-T sequence, such as TT, TTT, TTTT, TTTTT, TTTTTT, TTTTTTTT or TTTTTTTTTT. It is noted that spacers do not necessarily provide diversity, and as such, in a composition comprising single oligonucleotide species as described herein, some or all of the single oligonucleotide species may have the same spacer sequences. Optionally, all unique oligonucleotide species of the composition comprise the same spacer sequence.

Groups and crowding

Compositions comprising single oligonucleotide species as described in accordance with the compositions and methods of some embodiments herein may be arranged in spatially isolated groups, for example so that multiple samples can be analyzed, with one sample per group. In some embodiments, the unique oligonucleotide species are arranged in spatially isolated groups, each group comprising a plurality of unique oligonucleotides of the unique oligonucleotide species, such that the unique oligonucleotides of the same group comprise the same barcode sequence of sample, and such that different unique oligonucleotides from the same group comprise different molecular barcode sequences. As used herein, "spatial isolation" (and variations of this root term) means that the target nucleic acids of a sample can hybridize to the unique oligonucleotide species of the group without substantially cross-reacting with other groups, and without that the unique oligonucleotide species of the group hybridize substantially with target nucleic acids from other samples. As such, the sample barcode can identify which group a given single oligonucleotide species came from. Furthermore, after barcoding target nucleic acid sequences with unique oligonucleotide species, the sample barcode can identify which pool an encoded target nucleic acid sequence (or reverse transcription or amplicon thereof) comes from. .

In some embodiments, a substrate arranges the clusters such that they are spatially isolated from each other. For example, a multi-well plate can arrange spatially isolated groups, such that each group is in a separate well. For example, each group can be immobilized on a different bead. Optionally, each well of a multi-well plate may contain a single bead on which the pool of single oligonucleotide species is immobilized, such that a different pool of oligonucleotide species is placed in each well of the multi-well plate. unique.

In some embodiments, the unique oligonucleotide species are arranged in at least 2 groups, for example, at least 2, 3, 4, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 16, 17, 18, 19, 20, 21 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 72, 96, 99, 100, 110, 120, 144, 168, or 192, groups, including intervals between any two of the listed values. Optionally, there may be at least 100 unique oligonucleotides per group.

In some embodiments, the unique oligonucleotide species are arranged in at least 2 groups, and there are at least 100 unique oligonucleotides per group, for example, at least 100, 200, 300, 400, 500, 1000, 2000, 3000 , 4000, 5000, 6000, 6500, 10,000 or 65,000 species of unique oligonucleotides per group.

In some embodiments, the unique oligonucleotide species are arranged in at least 24 groups, and there are at least 100 unique oligonucleotides per group, for example, at least 100, 200, 300, 400, 500, 1000, 2000, 3000 , 4000, 5000, 6000, 6500, 10,000 or 65,000 species of unique oligonucleotides per group.

In some embodiments, the unique oligonucleotide species are arranged in at least 48 groups, and there are at least 100 unique oligonucleotides per group, for example, at least 100, 200, 300, 400, 500, 1000, 2000, 3000 , 4000, 5000, 6000, 6500, 10,000 or 65,000 species of unique oligonucleotides per group.

In some embodiments, the unique oligonucleotide species are arranged in at least 96 groups, and there are at least 100 unique oligonucleotides per group, for example, at least 100, 200, 300, 400, 500, 1000, 2000, 3000 , 4000, 5000, 6000, 6500, 10,000 or 65,000 species of unique oligonucleotides per group.

Substrates

Single oligonucleotide species according to the compositions and methods of some embodiments herein may be immobilized on substrates, for example, beads, wells of multi-well plates, or arrays. For example, unique oligonucleotide species that have the same barcode sample, but different molecular barcodes can be immobilized on a substrate, such as on a single bead, or in a single well of a multi-well plate. As such, when immobilized single oligonucleotide species from a particular substrate are contacted with a particular sample, such as an individual cell, the immobilized single oligonucleotide species, immobilized on that substrate will associate with target nucleic acids from the same sample. If single oligonucleotide species immobilized on the same substrate have the same sample barcode, while those on other substrates have different sample barcodes, the sample associated with each target nucleic acid can be easily identified (e.g., all single oligonucleotide species from substrate #1 have sample barcode #1, all unique oligonucleotide species from substrate #2 have sample barcode #2).

In some embodiments, such configurations in which single oligonucleotide species are immobilized on a substrate may be useful to facilitate efficiency and performance. For example, individual cells can be added to the wells of a multi-well plate such that there is no more than one individual cell in each well. If the unique oligonucleotide species immobilized in a given well (e.g., in the well or on a bead) comprise a unique sample barcode, the barcoded target nucleic acids and the reverse transcripts and amplicons thereof corresponding to the individual cell can be identified and quantified, even if barcoded reverse transcripts or amplicons from multiple samples are pooled.

A substrate may comprise a type of solid support. A substrate may refer to a continuous solid or semi-solid surface on which the methods of the disclosure may be performed. A substrate can refer to an array, a cartridge, a chip, a device, and a slide, for example. As such, "solid support" and "substrate" can be used interchangeably.

A solid substrate or support according to some embodiments herein may encompass any type of solid, porous or hollow sphere, ball, bearing, cylinder or other similar configuration composed of plastic, ceramic, metal or polymeric material (e.g., hydrogel). on which a nucleic acid can be immobilized (for example, covalently or non-covalently). A solid substrate or support may comprise a discrete particle that may be spherical (e.g., microspheres) or have a non-spherical or irregular shape, such as cubic, cuboid, pyramidal, cylindrical, conical, oblong or disc-shaped, and the like. A plurality of solid supports spaced in an array may not comprise a substrate. A solid support can be used interchangeably with the term "pearl."

In some embodiments, a plurality of substrates are provided, and the diversity of sample barcodes may be represented on the plurality of substrates, such that unique substrates may comprise unique sample barcodes.

Barcode coding methods

According to some embodiments herein, methods of specifically barcoding nucleic acids from two or more samples are described. Each sample may comprise nucleic acids. The method may comprise contacting each sample with a pool comprising a plurality of single oligonucleotide species, as described herein. Each sample can be contacted in spatial isolation from the other samples. The unique polynucleotide species of each group may comprise the same sample barcode and comprise different molecular barcodes. The method may include hybridization of specific target regions of at least some oligonucleotides of the single oligonucleotide species with at least some of the nucleic acids of the sample. The method may include extending the hybridized oligonucleotides to produce chains comprising an oligonucleotide of the single oligonucleotide species and a sequence complementary to the target region. Therefore, for each sample, the chains produced may comprise the same sample barcode and different molecular barcodes. For different samples, the sample barcodes may be different. In some embodiments, the single oligonucleotide species contacted with the sample consist or essentially consist of single oligonucleotide species in which each molecular barcode has a G content of less than 50%. In some embodiments, the molecular barcodes of all single oligonucleotide species collectively have a G content of no more than 12.5%. Optionally, each group comprises at least 100 unique oligonucleotide species, for example at least 100, 500, 100, 500, 1000, 2000, 6500, or 65,000 unique oligonucleotide species. Such methods, according to some embodiments herein, may barcode nucleic acids from two or more different samples, such as a single cell sample, to allow analysis and quantification of nucleic acids from a single cell. cell.

Optionally, each sample is contacted with a single pool of unique oligonucleotide species in spatial isolation from the other pools and samples. In some embodiments, the groups are spatially isolated by being in unique contained spaces, for example, different wells of a multi-well plate, different test tubes, different microfluidic channels, or the like. In some embodiments, the groups are spatially isolated by being in different regions of a surface, for example reaction points in a matrix.

The oligonucleotides can be contacted with the target nucleic acids of the sample under standard hybridization conditions (for example, in standard buffers and at a temperature below the Tm of a portion of the uniform region that is complementary to the target nucleic acid). Optionally, for example if the target nucleic acids comprise ^Rn , the target nucleic acids of the sample are reverse transcribed after hybridization to the single oligonucleotide species, to generate DNA ( ^for example cDNA). Optionally, the products of the hybridization or reverse transcription reaction are amplified to generate a DNA library. Amplification can be carried out by PCR under standard conditions, or by other suitable methods, for example isothermal amplification, rolling circle amplification, and the like. Optionally, the amplification may comprise the amplification of reverse transcripts or hybridized nucleic acids with a polymerase with 5' to 3' activity. Optionally, the amplification products can be analyzed further, for example by sequence. Optionally, reverse transcripts or amplicons from different pools can be pooled for sequencing, as different sample barcodes will indicate the corresponding pool (and sample) of each individual barcoded nucleic acid. It should be noted that there may be a bias in favor of G-rich molecular barcoding or single oligonucleotide species, and methods according to some embodiments herein may minimize or eliminate such bias. As an example, when quantifying nucleic acids from the sample at the single nucleic acid level, it may be useful to minimize or eliminate such bias that could favor the representation of some nucleic acids and bias the quantitative results in favor of certain types of codes. bars instead of representative target nucleic acids from the sample. In some embodiments, methods comprising single oligonucleotide species, as described herein, reduce bias by increasing the sensitivity, reducing the relative standard error, or by increasing the sensitivity and reducing the standard error.

Optionally, the method may further comprise determining the nucleic acid sequences of the chains comprising the oligonucleotides of the single oligonucleotide species and the sequence complementary to the target. For example, reverse transcripts, amplicons, and/or cDNA libraries generated from hybridization and extension of single oligonucleotide probes can be sequenced. Any suitable sequencing method known in the art, preferably high-throughput approaches, may be employed. For example, cycle array sequencing can also be used using platforms such as Roche 454, Illumina Solexa, ABI-SOLiD, ION Torrent, Complete Genomics, Pacific Bioscience, Helicos, or the Polonator platform. Sequencing may include MiSeq sequencing. Sequencing may include HiSeq sequencing. If appropriate, single oligonucleotide species may include adapters to facilitate high-throughput sequencing, for example universal priming sites. Optionally, barcoded nucleic acids corresponding to two or more different samples (e.g., from two or more different groups) can be pooled or combined for sequencing. Optionally, all groups are combined or pooled for sequencing. Without being limited by any theory, it is noted that the sample barcodes can identify the corresponding sample (or group) of the nucleic acids encoded with combined barcodes, such combination or grouping of the nucleic acids can facilitate the performance and /or utilization of resources for sequencing, and accurate barcoding according to some embodiments herein can minimize or eliminate bias, even when analyzing a large number of different nucleic acids.

The barcode region of each group comprises a sample barcode comprising at least 3 nucleotides as described herein, for example at least 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 , 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides, including intervals between any two of the values listed. Optionally, the sample barcode is located 3' of the molecular barcode. Optionally, the molecular barcode is located 3' of the sample barcode. Optionally, the single oligonucleotide species comprise the same sample barcode, which may be a different sample barcode from the single oligonucleotide species of other groups. Optionally, the sample barcode has a low G content as described herein, for example, less than 50% G, for example, less than 45% G, 40% G, 35% G, 30% G, 25% G, 20% G, 15% G, 12.5% G, 10% G, 7.5% G, 5% G or 2.5% of G. Optionally, at least 95%, 99% or 99.9% of the barcodes in the sample have a low G content as described herein. Optionally, each unique oligonucleotide comprises a spacer 3' of the molecular barcode and 5' of the uniform region as described herein.

The single oligonucleotide species contacted with the sample essentially consist of single oligonucleotide species in which each molecular barcode has a G content of less than 50% as described herein, for example, less than 45% of G, 40% of G, 35% of G, 30% of G, 25% of G, 20% of G, 15% of G, 12.5% of G, 10% of G, 7.5% of G, 5% G, or 2.5% G, or 0% G. In some embodiments, all molecular barcodes of the unique oligonucleotide species in contact with the sample comprise molecular barcodes having a G content of less than 50% as described herein, for example, less than 45% G, 40% G, 35% G, 30% G, 25% G, 20% G, 15% G, 12.5% G, 10% G, 7.5% G, 5% G, or 2.5% G content, or 0% G content.

In some embodiments, the molecular barcodes of all unique oligonucleotide species collectively have a G content of less than 12.5% G, 10% G, 7.5% G, 5% G, or 2.5% G, or 0% G, including intervals between any two of the listed values.

In some embodiments, each unique oligonucleotide species further comprises a uniform region 3' of the barcode region. The uniform region comprises at least 10 nucleotides complementary to a target nucleic acid sequence (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides complementary to the target, including intervals between any two of the values listed), and is configured for amplification from 5' to 3' of the nucleic acid sequence aim. Optionally, the uniform region comprises an oligo dT sequence.

In some embodiments, the single oligonucleotide species of each group are immobilized on a substrate as described herein, such that the single oligonucleotide species immobilized on a given substrate comprise the same sample barcode, and different Unique oligonucleotide species immobilized on the substrate comprise different molecular barcodes. By way of example, substrates may include wells in multi-well plates (e.g., 24-, 48-, and 96-well plates), spots in an array, beads, and the like. In some embodiments, at least 100 unique oligonucleotides are immobilized on each substrate. Optionally, all single oligonucleotide species immobilized on a given substrate (and therefore in the same pool) comprise the same sample barcode. In some embodiments, each sample barcode has a G content of 50% or less, for example 50% or less, 40% or less, 25% or less, 20% or less, 12.5% or less, 10% or less, or 5% or less.

In some embodiments, the molecular barcodes of the single oligonucleotide species collectively have a G content of less than 12.5%, for example less than 12.5%, less than 10%, less than 7.5%, less than 5%, less than 2.5% or less than 1%.

In some embodiments, the barcode regions of the single oligonucleotide species collectively have a G content of no more than 12.5%, for example no more than 12.5%, no more than 10%, not more than 7.5%, not more than 5%, not more than 2.5%, or not more than 1%.

In some embodiments, for at least 95% of the unique oligonucleotide species, any G in the molecular barcode is not adjacent to another G. For example, for at least 95%, 96%, 97%, For 98%, 99%, 99.5%, 99.9%, or 99.99%, of the unique oligonucleotide species, any G in the molecular barcode may be non-adjacent to another G.

In some embodiments, each group consists of, or essentially consists of, single oligonucleotide species for which any G of the molecular barcode is not adjacent to another G.

In some embodiments, at least 95% of the molecular barcodes of the single oligonucleotide species comprise a sequence that sums to at least 6 alternating H and N, for example at least 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 alternating H and N, including intervals between any two of the listed values. As an example, 6 alternating H and N can be represented as HNHNHN. It should be noted that each H can be the same or different from any other H, and that each N can be the same or different from any other N.

In some embodiments, at least 95% of the molecular barcodes of the single oligonucleotide species comprise the sequence HNHNHNHN, in which each "H" is any one of A, C, or T, and in which each "N" is any one of A, G, C, or T. For example, at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 99 .99% of the molecular barcodes of the single oligonucleotide species can comprise the sequence HNHNHNHN. It is noted that each H can be the same or different from any other H, and that each N can be the same or different from any other N. In some embodiments, each molecular barcode of the unique oligonucleotide species comprises the sequence HNHNHNHN.

In some embodiments, at least 99% of the molecular barcodes of the single oligonucleotide species comprise the sequence HHHHHHHH, where each "H" is any of A, C or T. For example, at least the 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 99.99% of the molecular barcodes of the single oligonucleotide species may comprise the sequence HHHHHHHH. In some embodiments, each molecular barcode of the unique oligonucleotide species comprises the sequence HHHHHHHH.

In some embodiments, each unique oligonucleotide comprises a spacer 3' to the barcode region and 5' to the target-specific region. Optionally, the spacer may comprise the sequence HNH, in which each "H" is any of A, C, or T, and in which each "N" is any of A, G, C, or T. Optionally, the spacer may comprise the sequence HHH. Optionally, the spacer may comprise the sequence HNHNHNHN. Optionally, the spacer may comprise the sequence HHHHHHHH. It is noted that each H can be the same or different from any other H, and that each N can be the same or different from any other N. In some embodiments, each single oligonucleotide comprises a spacer 3 'of the barcode region and 5 ' of the specific region of the target, said spacer comprising the sequence HNHNHNHN, in which each "H" is any of A, C, or T, and wherein each "N" is any of A, G, C, or T. In some embodiments, the single oligonucleotide comprises a spacer 3' of the barcode region and 5' of the target-specific region, said spacer comprising the sequence HHHHHHHH, in which each "H" is any of A, C or T.

In some embodiments, at least one group comprises at least two oligonucleotides of the same single oligonucleotide species. In some embodiments, no group comprises two oligonucleotides of the same single oligonucleotide species.

In some embodiments, the target specific region may comprise a sequence for 5' to 3' amplification of a target nucleic acid. In some embodiments, the target-specific region comprises an oligo dT sequence. In some embodiments, the target-specific region comprises a sequence flanking a sequence encoding a variable region of an immune cell receptor or immunoglobulin.

In some embodiments, for each unique oligonucleotide species, the molecular barcode is 3' to the sample barcode. In some embodiments, for each unique oligonucleotide species, the sample barcode is 3' to the molecular barcode.

In some embodiments, each unique oligonucleotide species has a length of at least 24 nucleotides. In some embodiments, each unique oligonucleotide species has a length of 24-140 nucleotides.

In some embodiments, each pool comprises at least 1000 unique oligonucleotide species. In some embodiments, each pool comprises at least 6,500 unique oligonucleotide species. In some embodiments, each pool comprises at least 65,000 unique oligonucleotide species.

In some embodiments, at least 48 unique samples are each contacted with a different set of unique oligonucleotide species. For example, at least 48, 72, 96, 120, 144, 168 or 192 samples can contact a different group. For example, each sample may be contacted with a different set of unique oligonucleotide species in one well of a multi-well plate.

In some embodiments, at least 99% of the samples comprise no more than one cell each. For example, at least 99%, 99.5%, 99.9% or 99.99% of the samples may comprise no more than one cell. By way of example, a solution comprising multiple cells may be diluted to a suitable concentration such that there is a suitably high probability that each sample does not comprise more than one cell.

In some embodiments, the single oligonucleotide species of each group are immobilized on a substrate, such that the sample barcodes but not the molecular barcodes are the same for the single oligonucleotide species immobilized on each substrate. As such, each group can be identified by a particular sample barcode. It should be noted that different substrates (and as such, different groups) can be associated with different sample barcodes. In some embodiments, the substrate comprises a spatially isolated region of a surface, such that substrates of different groups comprise different spatially isolated regions of the surface. In some embodiments, the substrate comprises one well of a multi-well plate. In some embodiments, the substrate comprises a bead.

In some embodiments, the single oligonucleotide species further comprises an adapter configured to immobilize the single oligonucleotide to the substrate, wherein said barcode region is located 3' of the adapter. Optionally, each adapter may comprise a universal priming site, for example for use in sequencing.

Methods of making compositions comprising single oligonucleotide species

According to some embodiments herein, methods for making a composition comprising single oligonucleotides are described. The method may comprise providing a plurality of different sample barcodes as described herein. The method may comprise providing a plurality of different molecular barcodes as described herein. The method may comprise synthesizing a plurality of single oligonucleotide species as described herein, wherein each single oligonucleotide species comprises a barcode region comprising a sample barcode and a molecular barcode as described at the moment. The method may comprise arranging the plurality of unique oligonucleotide species into spatially isolated groups. Each group may comprise multiple species of unique oligonucleotides, such that species of unique oligonucleotides from the same group comprise the same sample barcode sequence, and such that different species of unique oligonucleotides from the same group comprise different sequences of molecular barcode. Optionally, the unique oligonucleotide species are arranged in the spatially isolated pools concurrently with the synthesis. Optionally, the single oligonucleotide species are arranged in the groups spatially isolated after synthesis. Optionally, the composition consists essentially of single oligonucleotide species in which each molecular barcode has a G content of less than 50%. Optionally, the molecular barcodes of all single oligonucleotide species collectively have a G content of no more than 12.5%. Optionally, the composition consists essentially of single oligonucleotide species in which each molecular barcode has a G content of less than 50 and the molecular barcodes of all single oligonucleotide species collectively have a G content of no more of 12.5%. In some embodiments, each spatially isolated group comprises at least 100 unique oligonucleotide species, for example, at least 100, 200, 300, 400, 500, 1000, 2000, 3000, 5000, 6500 or 65,000 unique oligonucleotide species. In some embodiments, each spatially isolated group comprises at least 1000 unique oligonucleotide species.

Single oligonucleotides can be synthesized using any of the suitable methods. In some embodiments, the sample barcode sequences and the molecular barcode sequences are generated in silico, and unique oligonucleotide species comprising the sample barcode sequences and the molecular barcode sequences are synthesized. molecular rods together, for example using chemical synthesis of oligonucleotides. In some embodiments, a plurality of oligonucleotides comprising the same sample barcode sequence are pooled in spatial isolation, and ligated to a plurality of oligonucleotides comprising molecular barcodes, for example by hybridization and extension, or by ligation. . Such an approach can be performed in multiple spatially isolated environments in parallel, or sequentially, to achieve multiple species of unique oligonucleotides.

Optionally, each unique oligonucleotide species further comprises a uniform region 3' of the barcode region, as described herein. Optionally, the uniform region comprises a specific region of the target. In some embodiments, the target specific region comprises at least 10 nucleotides complementary to a target nucleic acid (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides complementary to the target, including intervals between any two of the values listed), and is configured for amplification from 5' to 3' of the target nucleic acid sequence. Optionally, the uniform region comprises an oligo dT sequence.

In some embodiments, the plurality of molecular barcodes (and as such, the plurality of single oligonucleotide species) essentially consists of single oligonucleotide species in which each molecular barcode has a G content of no more than 50%. as described herein, for example, less than 50% G, 45% G, 40% G, 35% G, 30% G, 25% G, 20% G, 15% G, 12.5% G, 10% G, 7.5% G, 5% G, or 2.5% G, or 0% G. In some embodiments, all molecular barcodes of the single oligonucleotide species have molecular barcodes that have a G content of no more than 50% as described herein. In some embodiments, all molecular barcodes of the single oligonucleotide species have molecular barcodes that have a G content of no more than 50% as described herein, for example, less than 50% G, 45% G, 40% G, 35% G, 30% G, 25% G, 20% G, 15% G, 12.5% G, 10% G, 7.5 % G, 5% G, or 2.5% G, or 0% G content. In some embodiments, each molecular barcode of each single oligonucleotide species has a G content of no more than 50%, for example, less than 50% G, 45% G, 40% G, 35% G, 30% of G, 25% of G, 20% of G, 15% of G, 12.5% of G, 10% of G, 7.5% of G, 5% of G, or 2.5 % G, or 0% G.

In some embodiments, the molecular barcodes of all single oligonucleotide species in the plurality of single oligonucleotide species collectively have a G content of less than 12.5% G, 10% G, 7.5% G, 5% of G, or 2.5% of G, or 0% of G, including intervals between any two of the values listed.

In some embodiments, each sample barcode has a G content of 50% or less, as described herein, for example, less than 50%, 45% G, 40% G, 35% G , 30% of G, 25% of G, 20% of G, 15% of G, 12.5% of G, 10% of G, 7.5% of G, 5% of G, or 2.5% of G content, or 0% G content.

In some embodiments, the sample barcodes of all single oligonucleotide species in the plurality of single oligonucleotide species collectively have a G content of no more than 50% G, e.g., less than 50%, 45% of G, 40% of G, 35% of G, 30% of G, 25% of G, 20% of G, 15% of G, 12.5% of G, 10% of G, 7.5% of G, 5% of G, or 2.5% of G, or 0% of G, including intervals between any two of the values listed. In some embodiments, the sample barcodes of all single oligonucleotide species of the plurality of single oligonucleotide species collectively have a G content of no more than 12.5%.

In some embodiments, for at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 99.99%) of the single oligonucleotide species, any G in the molecular barcode is not adjacent to another G. In some embodiments, the plurality of single oligonucleotide species essentially consists of single oligonucleotide species for which any G in the molecular barcode is not adjacent to another G.

In some embodiments, at least 95% of the molecular barcodes of the single oligonucleotide species comprise the sequence HNHNHNHN, wherein each "H" is any of A, C, or T, and wherein each " N" is any of A, G, C, or T. For example, at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 99.99% of the molecular barcodes of the single oligonucleotide species may comprise the sequence HNHNHNHN. It is noted that each H can be the same or different from any other H, and that each N can be the same or different from any other N. In some embodiments, each molecular barcode of the unique oligonucleotide species comprises the sequence HNHNHNHN.

In some embodiments, at least 99% of the molecular barcodes of the single oligonucleotide species comprise molecular barcodes comprising the sequence HHHHHHHH, where each "H" is any of A, C or T. For For example, at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 99.99% of the molecular barcodes of the single oligonucleotide species may comprise the sequence HHHHHHHH. In some embodiments, each molecular barcode of the unique oligonucleotide species comprises the sequence HHHHHHHH.

In some embodiments, each single oligonucleotide species comprises a spacer 3' to the barcode region and 5' to the target-specific region. Optionally, the spacer may comprise the sequence HNH, in which each "H" is any of A, C, or T, and in which each "N" is any of A, G, C, or T. Optionally, the spacer may comprise the sequence HHH. Optionally, the spacer may comprise the sequence HNHNHNHN. Optionally, the spacer may comprise the sequence HHHHHHHH. It should be noted that each H may be the same as or different from any other H, and that each N may be the same as or different from any other N. In some embodiments, each single oligonucleotide species comprises a spacer 3' of the barcode region. and 5' of the target-specific region, said spacer comprising the sequence HNHNHNHN, in which each "H" is any of A, C or T, and in which each "N" is any of A, G, C or T. In some embodiments, the single oligonucleotide comprises a spacer 3' of the barcode region and 5' of the target-specific region, the spacer comprising the sequence HHHHHHHH, wherein each "H" is any of A, C or T.

In some embodiments, at least one group comprises at least two oligonucleotides of the same single oligonucleotide species. In some embodiments, no group comprises two oligonucleotides of the same single oligonucleotide species.

In some embodiments, the target specific region may comprise a sequence for 5' to 3' amplification of a target nucleic acid. In some embodiments, the target-specific region comprises an oligo dT sequence. In some embodiments, the target-specific region comprises a sequence flanking a sequence encoding a variable region of an immune cell receptor or immunoglobulin.

In some embodiments, for each unique oligonucleotide species, the molecular barcode is 3' to the sample barcode. In some embodiments, for each unique oligonucleotide species, the sample barcode is 3' to the molecular barcode.

In some embodiments, each unique oligonucleotide species has a length of at least 24 nucleotides. In some embodiments, each unique oligonucleotide species has a length of 24-140 nucleotides.

In some embodiments, each pool comprises at least 6,500 unique oligonucleotide species. In some embodiments, each pool comprises at least 65,000 unique oligonucleotide species.

In some embodiments, the method further comprises immobilizing the unique oligonucleotide species of each spatially distinct group on a substrate, such that the sample barcodes but not the molecular barcodes are the same for the unique oligonucleotide species. immobilized on each substrate. As such, each group can be identified by a particular sample barcode. It should be noted that different substrates (and as such, different groups) can be associated with different sample barcodes. In some embodiments, the substrate comprises a discrete region of a surface, such that the substrates of the different groups comprise the different discrete regions of the surface. In some embodiments, the substrate comprises one well of a multi-well plate. In some embodiments, the substrate comprises a bead. In some embodiments, the single oligonucleotide species are immobilized on the substrate via a covalent bond. In some embodiments, the single oligonucleotide species are immobilized on the substrate by a magnetic or electromagnetic force. In some embodiments, the single oligonucleotide species are embedded in the substrate, to immobilize them on the substrate.

In some embodiments, at least 48 spatially distinct groups are made, for example at least 48, 72, 96, 120, 144, 168 or 192 spatially distinct groups.

In some embodiments, the single oligonucleotide species are arranged in groups spatially isolated simultaneously with said synthesis. In some embodiments, the single oligonucleotide species are arranged in spatially isolated groups after said synthesis.

Oligonucleotides

Outside the scope of the invention, an oligonucleotide comprising a barcode region 3' of the adapter region is described. The barcode region may comprise a molecular barcode as described herein, wherein the molecular barcode has a G content of no more than 50%. The molecular barcode may comprise at least 7 nucleotides.

In some embodiments, the oligonucleotide further comprises a sample barcode as described herein. The sample barcode may comprise at least 3 nucleotides.

In some embodiments, the oligonucleotide further comprises a uniform region 3' of the barcode region. In some embodiments, the uniform region comprises a target-specific region comprising at least 10 nucleotides complementary to a target nucleic acid as described herein. In some embodiments, the uniform region comprises an oligo dT sequence. In some embodiments, the oligonucleotide further comprises an adapter region 5' of the barcode region. In some embodiments, the oligonucleotide further comprises a spacer 3' to the barcode region and 5' to the uniform region as described herein.

Alternative embodiments

Designs are provided for accurate and unbiased barcoding of nucleic acid transcripts. These primer designs are composed of nucleic acid (DNA, RNA or LNA) sequences that can target specific nucleic acid (DNA/RNA) transcripts through a gene-specific approach, or target a large set of transcripts. nucleic acids (for example, through the poly-A tail of messenger RNAs or other consensus sequences) (FIGS. 1A-C). Along with the target sequences, these primers include two categories of nucleic acid barcodes: 1) molecular barcodes, also called "molecular barcodes", Molecular Index (MI) or Unique Molecular Identifier (UMI) and 2) sample barcodes, also called Sample Index (SI) (FIGS. 1A-C). The MI and SI barcodes are used to uniquely mark for the identification of its original target transcript through the MI, and its sample origin (SI) after library preparation for next generation sequencing or others. sequence reading and barcode reading methods. Some embodiments detail different MI and SI arrangements within the primer that ensure unbiased and random labeling of barcodes to target transcripts for accurate gene expression profiling (see, for example, FIGS. 3A- 3H) (see Fu et al. 2011 PNAS 108: 9026 9032).

In some embodiments, single primer designs can reduce "barcode bias", which is a phenomenon seen in prior art designs, so with high G nucleotide barcodes. it is used more often compared to other barcode compositions (FIG. 4). In certain cases where conventional primers are used to target and label mRNA transcripts using a gene-specific sequence, IMs with high % G content and multiple Gs are preferentially observed (FIG. 4). Furthermore, without being limited by any theory, in conventional designs, SIs associated with non-target genes observe small amounts of PCR errors because these primers cross-react during a sample multiplex PCR (FIGS. 4-5). . These cross-reactive events often occur with primers with high % G content and multiple Gs, possibly due to a unique secondary structure called "G-Quaduplexes" in G-rich oligonucleotides. Because these MI/SI primers G-rich can form inter- and intramolecular complexes with each other, conventional DNA cleanup steps to remove primers (e.g., Ampure XP beads) may not be sufficient. To minimize these events from occurring, this invention involves providing several designs that reduce the G content of primers to minimize "barcoding bias" and primer crossover (FIGS. 3A-3H).

FIGS. 3A-3H show the state-of-the-art barcode design (FIG. 3A) and the novel designs to minimize bias as described (FIGS. 3B-H). In FIG. 3A, the conventional design allows MI composition of 'BBBBBBV' or 'NNNNNNNN' (B is C, G, or T; V is A, C, or G; N is A, T, G, or C). A subset of the MI barcode from the above design would be G-rich. In panel 3B-C, addition of TTT or TTTTT after the MI to reduce G-richness across the region (the length of the spacers does not G may be variable, and may be any non-G nucleotide). In FIG. 3D, the positions of SI and MI are swapped, such that G-rich MIs are not adjacent to potentially G-rich target regions. In FIGA. 3E-F, TTT or TTTTT is added to the design of FIG. 3D to further reduce the G-rich regions (note that the SIs are not G-rich, and the length of the non-G spacers can be variable and can be any non-G nucleotide). In the designs of FIGS. 3G- H, MIs are designed to be non-G rich by using 'HHHHHHHH' or 'HNHNHNHN,' where H is A, C, or T.

The G-rich drawbacks of conventional designs are observed in the method using gene-specific targeting. Some embodiments include the use of the new primer designs in gene-specific target panels, such as T cell receptors (TCR), where the primers are used to target specific TCR sequences. Designs according to some embodiments herein may alleviate crosstalk of SI barcodes, thereby reducing the "noise" of a TCR molecular barcoding assay.

Example 1: Precise Analysis of Sample Target Nucleic Acids Using Single Oligonucleotide Species

96 unique oligonucleotide species formats were designed, the sequences of which are summarized in FIGS.

5A-5E (SEQ ID NO: 1-96). The single oligonucleotide species formats A01-A12 (SEQ ID NO: 1-12) were provided as controls, in which the molecular barcode ("2° be") was NNNNNNNNN. As such, the molecular barcodes of these control single oligonucleotide species did not have any restrictions or limits on G content. The single oligonucleotide species formats B01-B12 (SEQ ID NO: 13-24) comprised a spacer of the TTT sequence 3' of the molecular barcode ("2° be") and 5' of the uniform region ("5 to 3' recognition sequence"). The unique oligonucleotide species formats C01-C12 (SEQ ID NO: 25-36) comprised a spacer of the TTTTT sequence 3' of the molecular barcode ("2° be") and 5' of the uniform region ("sequence recognition 5 to 3'"). The unique oligonucleotide species formats D01-D12 (SEQ ID NO: 37-48) comprised a molecular barcode ("1° be") 5' of the sample barcode ("2° bc"). The unique oligonucleotide species formats E01-E12 (SEQ ID NO: 49-60) comprised a barcode molecule ("1° be") 5' of the sample barcode ("2° be") and a TTT sequence spacer 3' of the barcode region and 4' of the uniform region. The unique oligonucleotide species formats F01-F12 (SEQ ID NO: 61-72) comprised a molecular barcode ("1° be") 5' of the sample barcode ("2° be") and a spacer of the TTTTT sequence 3' of the barcode region and 4' of the uniform region. The unique oligonucleotide species formats G01-G12 (SEQ ID NO: 73-84) comprised molecular barcodes ("2° be") each comprising the sequence HHHHHHHH (in which each "H" is a A, C or T, and in which any two "H" nucleotides can be the same or different). The unique oligonucleotide species formats H01-H12 (SEQ ID NO: 85-96) comprised molecular barcodes ("2° be") each comprising the sequence HNHNHNHN (in which each "H" is a A, C or T, and in which any two "H" nucleotides can be the same or different, and in which each "N" can be A, G, C or T, and in which any two "N" nucleotides They can be the same or different).

Amplification and analysis of target T cell receptor nucleic acid sequences were performed as follows: the Precise™ TCR Encoding Plate (BD Cellular Research Inc.) was thawed at room temperature and briefly rotated to Collect the 5μl of reagents in each well. The plate was placed on ice in a 96-well rack and the seal was removed very carefully, avoiding disturbing the reagents in the wells. Cells were sorted or added directly into the 96-well scramble plate (1 cell per well). The plate was sealed and vortexed briefly (5-10 s) to mix the reagents and then centrifuged briefly (~1000 rpm x 10 s). Sorted sample plates can optionally be stored at -80°C until ready to start the Precise™ protocol. For reverse transcription, the plate was incubated at 65°C for 3 minutes, cooled to 4°C, and placed on ice for 5 minutes. The reaction master mix was as follows:

T l 1

5 μl of RT MM was pipetted into each well. Each plate was sealed and a reverse transcriptase program was run: 42°C, 30 min; 80C, 5 min; 4C break.

cDNA purification was performed using the following protocol: Spin the plate (1000 rpm x 10 s). Combine all reactions in a single 2 ml tube. Add 8ul of Precise DBP Mix into the combined cDNA tube. Vortex gently to get the buffer into the solution. Add an equivalent volume of AMPure XP beads to the tube containing the pooled RT reaction product (1X volume of pooled PCR product). Pipette up and down to mix the sample and beads. Incubate the bead mixture at room temperature for 5 minutes. Open the cap to ensure that the beads are not disturbed. Place the tube over a magnet and wait until the liquid and beads separate. The liquid should be completely transparent before removing it. This may take up to 5 min. Hold the tube over the magnet, carefully remove only the liquid and discard it. Keeping the tube in the magnetic holder, gently rinse the beads once with 2 ml of 70% EtOH/30% H ₂ O. Prepare a fresh EtOH solution each day to prevent evaporation. Care should be taken not to disturb or resuspend the beads during the rinsing step. Immediately remove all solution of residual EtOH with a pipette. While the tube is over the magnet, tap to collect as much EtOH as possible at the bottom of the tube. To elute the product from the beads, remove the tube from the magnet. Transfer 68 µL of elution buffer to the tube containing the beads. Moisten the beads with the elution buffer by tilting the tube. Vortex well to mix and wait 1 minute. Replace the tube on the magnet. Wait until the solution clears, usually <5 min. Recover the purified solution of the PCR product by carefully pipetting the solution to separate it from the beads. Transfer the clear liquid product (68 µL) to a new 1.5 mL tube.

N1 PCR amplification (target amplification) was performed using the following protocol: Prepare the PCR mix (200ul) in the preamplification zone according to Table 2:

Divide this 200ul reaction mixture into 4 PCR tubes. Run 20 cycles of the following PCR conditions: 3 minutes annealing at 60C, 1 minute extension at 68C (approximately 2 hours). Combine all PCR products in a sample tube. Add 160ul of Ampure beads (0.8X), mix well with a pipette and incubate for 5 minutes at RT. Place the tube over a magnet and wait until the liquid and beads separate. The liquid should be completely transparent before removing it. This may take up to 5 min. Remove only the liquid and discard it. Gently rinse the tube once while on the magnetic holder with 1 ml of 70% EtOH/30% H ₂ O. Do not resuspend the beads during the rinsing step. Immediately remove all residual EtOH with a pipette. While the tube is over the magnet, tap to collect as much EtOH as possible at the bottom of the tube. Elute the PCR product in 50ul of elution buffer.

N1 PCR amplification (adapter and indexing PCR) was performed following the following protocol: Prepare the PCR mix (50ul) according to Table 3:

T l

Run 25 cycles of the following PCR conditions: 3 minutes annealing at 60C, 1 minute extension time at 68C. Purify at 0.8X Ampure twice, elute in 30 ul elution buffer.

FIG. 6 shows error correction for averaging sequence results (per individual TCRa CDR3). In FIG. 6, the Y axis represents the number of different molecular barcodes detected for TCRa.

Results are shown for reactions with 50 μg of target RNA (denoted as “50” in FIG. 6, e.g., A50, B50, C50, D50, E50, F50, G50) and negative controls receiving 0 μg of RNA. target (denoted as "0" in FIG. 6, for example, A0, B0, C0, ^d 0, E0, F0, G0). As such, 0 μg serves as a negative control and detector for assay noise leaking from the 50 μg sample. For reactions receiving target RNA, single oligonucleotide designs according to some embodiments herein B01-B12 ("B50"), C01-C12 ("C50"), D01-D12 ("D50"), E01- E12 ("E50"), F01-F12 ("F50"), G01-G12 ("G50") and H01-H12 ("H50") had lower error rates than conventional A01-A12 ("A50") formats. ). In particular, for the TRAC in this experiment, the “H” format had the highest sensitivity with low noise and performed better than the original barcoded primers (A).

Thus, compositions comprising single oligonucleotide species comprising barcode regions, and molecular barcodes having a relatively low G content, and/or single oligonucleotides comprising a 3' spacer of the molecular barcode and 5' of the uniform region, as described herein, produced amplification with a lower error rate, and therefore greater precision, than compositions comprising general polynucleotide species that did not comprise such characteristics.

Although various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes and are not intended to be limiting; the true scope is indicated in the following claims.

One skilled in the art will appreciate that, for this and other processes and methods disclosed herein, the functions performed in the processes and methods may be implemented in a different order. Furthermore, the steps and operations described are only provided by way of example, and some of the steps and operations may be optional, combined into fewer steps and operations, or expanded into additional steps and operations without detracting from the essence of the disclosed embodiments.

With respect to the use of substantially any plural and/or singular terms herein, those skilled in the art may translate from plural to singular and/or from singular to plural as appropriate for the context and/or application. The various permutations of singular/plural may be expressly set forth herein for the sake of clarity.

Those skilled in the art will understand that, in general, the terms used herein, and especially in the accompanying claims (e.g., the bodies of the attached claims) are understood to be "open" terms (e.g., the term "which includes" should be interpreted as "including but not limited to", the term "having" should be interpreted as "having at least", the term "includes" should be interpreted as "includes but not limited to", etc.) . Those skilled in the art will further understand that if a specific number of an entered claim is intended, such intention will be explicitly stated in the claim, and in the absence of such statement no such intention exists. For example, as an aid to understanding, the following appended claims may contain the use of the introductory phrases "at least one" and "one or more" to introduce the enumerations of the claims. However, the use of such phrases should not be construed to mean that the introduction of a claim statement by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim statement to embodiments containing only one of said statements, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (for example, "a" and/or "one" should be interpreted to mean "at least one" or "one or more"); The same goes for the use of definite articles used to introduce claims. Furthermore, even if a specific number of an introduced claim statement is explicitly recited, those skilled in the art will recognize that such statement must be interpreted as meaning at least the number stated (for example, the simple enumeration of "two statements", without other modifiers, means at least two statements, or two or more statements). Furthermore, in those cases in which a convention analogous to "at least one of A, B and C, etc." is used, such construction is generally understood in the sense in which a person skilled in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B and C together, etc.). In those cases in which a convention analogous to "at least one of A, B or C, etc." is used, such construction is generally understood in the sense in which a person skilled in the art would understand the convention (e.g. For example, "a system that has at least one of A, B, or C" would include, but would not be limited to, systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B and C together, etc.). Those skilled in the art will understand that virtually any word and/or disjunctive phrase that presents two or more alternative terms, whether in the description, claims or drawings, should be understood to contemplate the possibilities of including one of the terms, any of them. the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

Furthermore, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also described in terms of any individual member or subgroup of members of the Markush group.

As one skilled in the art will understand, for any and all purposes, such as providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. . Any enumerated interval can easily be recognized as sufficiently descriptive and allows the same interval to be decomposed into at least equal halves, thirds, fourths, fifths, tenths, etc. By way of non-limiting example, each interval described herein can be easily divided into a lower third, a middle third and an upper third, etc. As will also be understood by one skilled in the art, all terms such as "up to", "at least" and the like include the aforementioned number and refer to intervals that may be further divided into subintervals, as discussed above. Finally, as one skilled in the art will understand, a range includes each individual member. So, for example, a group that has 1-3 cells refers to groups that have 1, 2 or 3 cells. Similarly, a group that has 1-5 cells refers to groups that have 1, 2, 3, 4, or 5 cells, and so on.

Claims (15)

1. A composition comprising at least 1000 unique oligonucleotide species, each unique oligonucleotide species comprising a barcode region and a uniform region, the barcode region comprising a molecular barcode comprising at least 7 nucleotides, wherein the uniform region is 3' of the barcode region, the uniform region comprising a target-specific region 3' of the barcode region, the target-specific region comprising at least 10 nucleotides complementary to an acid target nucleic, wherein the unique oligonucleotide species comprise different nucleic acid sequences in their barcode regions, and where: (a) the composition consists essentially of single oligonucleotide species where each molecular barcode has a G content of less than 50%; I (b) the molecular barcodes of all single oligonucleotide species in the composition collectively have a G content of no more than 12.5%, wherein the barcode region further comprises a sample barcode comprising at least 3 nucleotides. 2. The composition of claim 1, wherein the composition consists essentially of single oligonucleotide species wherein each molecular barcode has a G content of less than 50%. 3. The composition of claim 1 or 2, wherein the molecular barcodes of all unique oligonucleotide species of the composition collectively have a G content of no more than 12.5%. 4. The composition of any of claims 1-3, wherein the molecular barcodes of all unique oligonucleotide species in the composition collectively have a G content of 2.5%-12.5%, e.g. .5%-10%. 5. The composition of any of claims 1-4, wherein the unique oligonucleotide species are arranged in at least two spatially isolated groups, each group comprising at least 100 unique oligonucleotides of the unique oligonucleotide species, wherein unique oligonucleotides from the same group comprise the same sample barcode sequence, and wherein different unique oligonucleotides from the same group comprise different molecular barcode sequences. 6. The composition of any of claims 1-5, wherein for at least 95% of the unique oligonucleotide species, any G in the molecular barcode is not adjacent to another G. 7. The composition of any of claims 1-6, wherein the specific target region comprises an oligo dT sequence. 8. The composition of any of claims 1-7, wherein the composition comprises at least 6,500 unique oligonucleotide species. 9. The composition of any of claims 5-8, wherein the unique oligonucleotide species of each group are immobilized on a substrate, such that the sample barcodes but not the molecular barcodes are the same for the oligonucleotide species immobilized on each substrate. 10. The composition of any of claims 1-9, wherein the target-specific region comprises a sequence flanking an immune cell receptor or an immunoglobulin variable region coding sequence. 11. A method of specifically barcoding a plurality of nucleic acids from two or more samples, each sample comprising nucleic acids, comprising: contacting each sample with a pool comprising at least 100 unique oligonucleotide species, wherein each sample is contacted in spatial isolation from the other samples, each unique oligonucleotide species comprising a barcode region comprising: a molecular barcode comprising at least 7 nucleotides; a sample barcode comprising at least 3 nucleotides; and a uniform region 3' of the barcode region, the uniform region comprising a target-specific region 3' of the barcode region, the target-specific region comprising at least 10 nucleotides complementary to a target nucleic acid, wherein the unique polynucleotide species of each group comprise the same sample barcode and comprise different molecular barcodes, and where: (a) the single oligonucleotide species in contact with the sample essentially consist of single oligonucleotide species where each barcode molecule has a G content of less than 50%; I (b) the molecular barcodes of all single oligonucleotide species collectively have a G content of no more than 12.5%; hybridizing the target-specific regions of at least some oligonucleotides of the single oligonucleotide species with at least some of the nucleic acids of the sample; and extend the hybridized oligonucleotides, thereby producing chains comprising an oligonucleotide of the single oligonucleotide species and a sequence complementary to the target, wherein for each sample, the chains comprise the same sample barcode and different molecular barcodes , and where for different samples, the molecular barcodes are different. 12. The method of claim 11, wherein the molecular barcodes of all single oligonucleotide species collectively have a G content of no more than 12.5%. 13. The method of any of claims 11-12, wherein the at least 100 unique oligonucleotide species of each group are immobilized on a substrate, such that the unique oligonucleotide species immobilized on a given substrate comprise the same sample barcode, and different single oligonucleotide species immobilized on the substrate comprise different molecular barcodes. 14. A method for making a composition comprising single oligonucleotides, the method comprising: providing a plurality of different sample barcodes comprising at least 3 nucleotides each, providing a plurality of different molecular barcodes comprising at least 7 nucleotides each; synthesize a plurality of unique oligonucleotide species, each unique oligonucleotide species comprising a barcode region comprising: a sample barcode and a molecular barcode; and a uniform region 3' of the barcode region, the uniform region comprising a target-specific region 3' of the barcode region, the target-specific region comprising at least 10 nucleotides complementary to a target nucleic acid; and arranging the unique oligonucleotide species in spatially isolated groups, where each group comprises multiple unique oligonucleotide species, such that the unique oligonucleotide species of the same group comprise the same sample barcode sequence, and wherein different species of unique oligonucleotides from the same group comprise different molecular barcode sequences, wherein each group comprises at least 1000 species of unique oligonucleotides, where for each group: (a) single oligonucleotide species essentially consist of single oligonucleotide species in which each molecular barcode has a G content of less than 50%; I (b) The molecular barcodes of all single oligonucleotide species collectively have a G content of no more than 12.5%. 15. A kit that includes: a composition of any of claims 1-9, wherein the target-specific region comprises a sequence flanking an immune cell receptor or an immunoglobulin variable region coding sequence; and a primer configured to hybridize on an opposite side of the variable region as the target-specific region, and to hybridize to a strand complementary to a strand hybridized by the target-specific region, and is therefore configured to amplify the variable region along with the specific target region.